Novel sulfonamide compounds for inhibition of metastatic tumor growth

ABSTRACT

Therapeutic ureido-sulfonamide compositions having compounds with the formula 
       R-Q-Ar—SO 2 NH 2  
 
     are disclosed, which compounds selectively inhibit CAIX and CAXII, and which are effective to inhibit hypoxic tumor growth, suppress metastases, and impair and deplete cancer stem cells in mammals.

BACKGROUND

Field of Invention

The disclosure is in the field of novel sulfonamide compounds,particularly for use as inhibitors of carbonic anhydrase IX and XII, inthe treatment of hypoxic and metastatic cancer, and the depletion ofcancer stem cells in mammals.

Description of Related Art

Sixteen different α-carbonic anhydrase (CA) isoforms have been isolatedand characterized in mammals, where they play important physiologicalroles. Some of them are cytosolic (CAI, CAII, CAIII, CAVII, CAXIII),others are membrane-bound (CAIV, CAIX, CAXII, CAXIV and CAXV), CA VA andCA VB are mitochondrial, and CAVI is secreted in saliva and milk. Themammalian CAs were the first such enzymes isolated and studied in detail(Supuran, C T. Nat. Rev. Drug Discov. 2008, 7, p 168, Supuran, C. T.;Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336) and many of them areestablished therapeutic targets. The classical CA inhibitors are theprimary sulfonamides, RSO₂NH₂, which have been in clinical use for morethan 50 years as diuretics and antiglaucoma drugs. In fact there arearound 30 clinically used drugs (or agents in clinical development)belonging to the sulfonamide or sulfamate class which show CAsinhibitory activity (Supuran, C. (2008) Nature, Vol 7: 168-181) and someof which are established as diuretics and antiglaucoma agents.

It has recently emerged that CA inhibitors have potential as, interalia, anticancer drugs. However critical barriers to the design of CAinhibitors as therapeutic agents are related to the high number ofisoforms in humans (i.e., 16 CAs, of which 13 have catalytic activity),their rather diffuse localization in many tissues/organs, and the lackof isozyme selectivity of the presently available inhibitors of thesulfonamide/sulfamate class. In fact, among derivatives mentioned above,there are no compounds which selectively inhibit CA isoforms withtherapeutic value (inhibition data of 1-25 sulfonamide compounds againstall human (h) CA isoforms are provided in Supuran, C. (2008) Nature 7:168-181.

Isozymes CAIX and CAXII are predominantly found in tumor cells and showa restricted expression in normal tissues. A helpful overview of thelimitations of current CAI is provided in Poulsen, Expert Opin. Ther.Patents (2010) 20(6):795-806. It is clear from this review that the useof CAIX inhibitors for cancer and metastasis has not been demonstratedeffectively, due to the limitations of the compounds available.

Evidence that certain sulfonamide CAIX inhibitors may indeed showantitumor effects, has been only very recently published (Ahlskog, J. K.J.; Dumelin, C. E.; Trüssel, S.; Marlind, J.; Neri, D. Bioorg. Med.Chem. Lett. 2009, 19, 4851.) Certain compounds have been disclosed, forexample in Maresca et al., PCT publication WO2009089383. Selectivity isan issue, however, as inhibition of housekeeping carbonic anhydrases iscontraindicated.

SUMMARY OF THE INVENTION

Provided herein are pharmaceutical compositions for treating hypoxic andmetastatic cancer, for the impairment or destruction of cancer stemcells, or for the treatment of any cancer characterized by increasedexpression of CAIX or CAXII.

One embodiment provides a pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound of Formula (I)

R-Q-Ar—SO₂NH₂   Formula (I)

wherein,

R is an aryl, heteroaryl, aralkyl, alkyl or cycloalkyl group, with orwithout a substituent;

Q is -L(CH₂)_(n)—, wherein n=0, 1 or 2;

L is —NHC(X)NH—, —NHC(S)SNH—, —NHC(O)NHC(S)NH—, or —SO₂NH—;

X is O or S; and

Ar is a C₆-C₁₀ aryl or a heteroaryl that contains at least oneheteroatom of oxygen, nitrogen or sulphur.

In certain embodiments, the following compounds are expressly excluded:

-   4-[(anilinocarbonyl)amino]benzenesulfonamide;-   4-{[(Pentafluorophenyl)carbamoyl]amino}benzenesulfonamide;-   4-{[(4′-Acetylphenyl)carbamoyl]amino}benzenesulfonamide;-   4-{[(4′-Chlorophenyl)carbamoyl]amino}benzenesulfonamide; or-   4-{[([4′-(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide.

In specific embodiments, the compound of Formula (I) areureido-sulfonamides. Specifically, Q is —NHCONH—, Ar is phenyl, and R isPhCH₂, Ph₂CH, 4-FC₆H₄, 4-ClC₆H₄, 4-BrC₆H₄, C₆F₅, 2-MeOC₆H₄, 4-AcC₆H₄,2-i-PrC₆H₄, 4-i-PrC₆H₄, 4-n-BuC₆H₄, 4-n-BuOC₆H₄, 4-n-octyl-C₆H₄,4-NCC₆H₄, 2-NCC₆H₄, 4-PhOC₆H₄, 2-PhC₆H₄, 3-O₂NC₆H₄, 4-MeO-2-MeC₆H₃,cyclopentyl, indan-5-yl, 3,5-Me₂C₆H₃, 4-CF₃C₆H₄, or 3,5-(CF₃)₂C₆H₃.

Examples of the compounds of Formula (I) include:

-   4-{[(Benzylamino)carbonyl]amino}benzenesulfonamide (MST-102);-   4-{[(Benzhydrylamino)carbonyl]amino}benzenesulfonamide (MST-103);-   4-{[(4′-Fluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-104);-   4-{[(4′-Bromophenyl)carbamoyl]amino}benzenesulfonamide (MST-105);-   4-{[(Pentafluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-107);-   4-{[(2′-Methoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-108);-   4-{[(4′-Acetylphenyl)carbamoyl]amino}benzenesulfonamide (MST-109);-   4-{[(2′-iso-Propylphenyl)carbamoyl]amino}benzenesulfonamide    (MST-110);-   4-{[(4′-iso-Propylpheyl)carbamoyl]amino}benzenesulfonamide    (MST-111);-   4-{[(4′-n-Butylphenyl)carbamoyl]amino}benzenesulfonamide (MST-112);-   4-{[(4′-Butoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-113);-   4-{[(4′-n-Octylphenyl)carbamoyl]amino}benzenesulfonamide (MST-114);-   4-{[(4′-Cyanophenyl)carbamoyl]amino}benzenesulfonamide (MST-115);-   4-{[(2′-Cyanophenyl)carbamoyl]amino}benzenesulfonamide (MST-116);-   4-{[(4′-Phenoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-117);-   4-{[(Biphenyl-2′-yl)carbamoyl]amino}benzenesulfonamide (MST-118);-   4-{[(3′-Nitrophenyl)carbamoyl]amino}benzenesulfonamide (MST-119);-   4-{[(4′-Methoxy-2′-methylphenyl)carbamoyl]amino}benzenesulfonamide    (MST-120);-   4-[(Cyclopentylcarbamoyl)amino]benzenesulfonamide (MST-122);-   4-{([(3′,5′-Dimethylphenyl)amino]carbonylamino)}benzenesulfonamide    (MST-123);-   4-{[(4′-Chlorophenyl)carbamoyl]amino}benzenesulfonamide (MST-124);-   4-{[(2′,3′-Dihydro-1H-inden-5′-ylamino]carbonylamino)}benzenesulfonamide    (MST-125);-   4-{[([4′-(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide    (MST-126);-   4-{[([3′,5′-bis(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide    (MST-127);-   3-(3-(4′-Iodophenyl)ureido)benzenesulfonamide (MST-128);-   3-(3-(4′-Fluorophenyl)ureido)benzenesulfonamide (MST-129);-   3-(3-(3′-Nitrophenyl)ureido)benzenesulfonamide (MST-130);-   3-(3-(4′-Acetylphenyl)ureido)benzenesulfonamide (MST-131);-   3-(3-(2′-Isopropylphenyl)ureido)benzenesulfonamide (MST-132);-   3-(3-(Perfluorophenyl)ureido)benzenesulfonamide (MST-133);-   4-(3-(4′-chloro-2-fluorophenyl)ureido)benzenesulfonamide (MST-134);-   4-(3-(4′-bromo-2′-fluorophenyl)ureido)benzenesulfonamide (MST-135);-   4-(3-(2′-fluoro-5′-nitrophenyl)ureido)benzenesulfonamide (MST-136);-   4-(3-(2′,4′,5′-trifluorophenyl)ureido)benzenesulfonamide (MST-137);-   4-(3-(2′-fluoro-5′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-138);-   4-(3-(2′-fluoro-3′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-139);-   4-(3-(2′,3′,4′-trifluorophenyl)ureido)benzenesulfonamide (MST-140);-   4-(3-(2′-fluorophenyl)ureido)benzenesulfonamide (MST-141);-   4-(3-(2′,4′-difluorophenyl)ureido)benzenesulfonamide (MST-142);-   4-(3-(3′-chlorophenyl)ureido)benzenesulfonamide (MST-143);-   4-(3-(2′,5′-dichlorophenyl)ureido)benzenesulfonamide (MST-144);-   4-(3-(2′-Chloro-5′-nitrophenyl)ureido)benzenesulfonamide (MST-145);-   4-(3-(2′-Chloro-4′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-146);-   4-(3-(2′,6′-difluorophenyl)ureido)benzenesulfonamide (MST-147); or-   4-(3-(perchlorophenyl)ureido)benzenesulfonamide (MST-148).

Further provided are methods of suppressing tumor growth, invasionand/or tumor metastases in a mammal by treating said mammal with thepharmaceutical compositions described herein.

Further provided is a method of reducing breast cancer cells number ormass in a mammal by treating said mammal with the pharmaceuticalcompositions described herein.

Also provided is a method of depleting cancer stem cells in a mammaliancancer stem cell population using the pharmaceutical compositionsdescribed herein.

Also provided is a method of inducing cell death in hypoxic cancer cellsusing the pharmaceutical compositions described herein.

A mammal so treated according to the present disclosure may be treatedwith additional chemotherapeutic or other anticancer agents. Any canceror tumor or cell population treated herein may express CAIX or CAXIIover and above the normal level for non-cancerous like-originatedtissues.

The tumors treated may be of the breast, lung, pancreatic, renal,prostate, cervical, colorectal cancer, or glioblastoma according tocertain embodiments. The use of the compositions to treat a mammalhaving cancer or a tumor may reduce or eliminate metastases.

The mammal may be human.

There is further provided a compound comprising formula (I)

R-Q-Ar—SO₂NH₂

wherein,

R is an aryl, heteroaryl, aralkyl, alkyl or cycloalkyl group, with orwithout a substituent;

Q is -L(CH₂)_(n)—, where n=0, 1 or 2;

L is —NHC(X)NH—, —NHC(S)SNH—, —NHC(O)NHC(S)NH—, or —SO₂NH—;

X is O or S; and

Ar is a C₆-C₁₀ aryl or a heteroaryl group that contains at least oneheteroatom of oxygen, nitrogen or sulphur.

In certain embodiments, the following compounds are expressly excluded:

-   4-[(anilinocarbonyl)amino]benzenesulfonamide;-   4-{[(Pentafluorophenyl)carbamoyl]amino}benzenesulfonamide;-   4-{[(4′-Acetylphenyl)carbamoyl]amino}benzenesulfonamide;-   4-{[(4′-Chlorophenyl)carbamoyl]amino}benzenesulfonamide;-   4-{[([4′-(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide;-   3-(3-(4′-Iodophenyl)ureido)benzenesulfonamide;-   3-(3-(4′-Fluorophenyl)ureido)benzenesulfonamide;-   3-(3-(3′-Nitrophenyl)ureido)benzenesulfonamide;-   3-(3-(4′-Acetylphenyl)ureido)benzenesulfonamide;-   3-(3-(2′-Isopropylphenyl)ureido)benzenesulfonamide; or-   3-(3-(Perfluorophenyl)ureido)benzenesulfonamide.

In certain embodiments, Q may be —NHCONH—, Ar is phenyl, and R may bePhCH₂, Ph₂CH, 4-FC₆H₄, 4-ClC₆H₄, 4-BrC₆H₄, C₆F₅, 2-MeOC₆H₄, 4-AcC₆H₄,2-i-PrC₆H₄, 4-i-PrC₆H₄, 4-n-BuC₆H₄, 4-n-BuOC₆H₄, 4-n-octyl-C₆H₄,4-NCC₆H₄, 2-NCC₆H₄, 4-PhOC₆H₄, 2-PhC₆H₄, 3-O₂NC₆H₄, 4-MeO-2-MeC₆H₃,Cyclopentyl, Indan-5-yl, 3,5-Me₂C₆H₃, 4-CF₃C₆H₄, or 3,5-(CF₃)₂C₆H₃.

Preferred compounds include the following:

The pharmaceutical compositions described herein are characterized inthat they inhibit the activity of tumor-related CAIX and CAXII to agreater degree than they inhibit the activity of CAI and CAII in vitro.

There are further provided compositions and their use to inhibitinvasion, and/or induce cell death of human breast cancer cells inhypoxia.

There are further provided compositions and their use to impairmaintenance of breast cancer stem cells through the inhibition of CAIXactivity.

There are further provided compositions and their use to deplete thecancer stem cell population in human breast cancer by inhibiting CAIXactivity.

There is also provided a method of treating metastatic or hypoxic cancerwith MST-017, MST-114, MST-119, MST-104, or MST-130.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows In Kaplan-Meier analyses of CAIX expression associationwith relapse free survival (A), distant relapse free survival (B) andbreast cancer specific survival (C), achieving very high levels ofstatistical significance (p<10-17, p<10-16, and p<10-13, respectively).The 10 year distant relapse free survival and breast cancer specificsurvival rates in the CAIX positive versus CAIX negative groups were 57%compared to 73%, and 62% compared to 78%, respectively. In multivariateanalyses, including all standard prognostic variables and biologicalsubtypes, CAIX expression remained a strong independent poor prognosticfactor with a hazard ratio of 1.4.

FIG. 2 shows (A) cell cultures for three cell lines 67NR, 66cl4 and 4T1together with mouse models demonstrating bioluminescent labeling of thetumor cells in vivo and (B) hypoxia-induced gene expression table forthe three tumor types (high expression, dark; low expression, light).

FIG. 3 shows a Western blot showing CAIX over-expression in the primarytumors. NMG=normal mammary gland. Beta-actin served as a loadingcontrol.

FIG. 4 shows (A) graph of the data relating to CAIX expression inmetastatic (4T1, 66cl4) and non-metastatic (67NR) cells incubated for 72h as measured by qRT-PCR (graph) and Western blot of lysates (gel). Dataare expressed as mean±s.e.m. n=3, **P<0.005, ***P<10⁻³. Beta-actin isshown as a loading control. (B) 4T1 cells expressing non-silencing shRNA(shNS) or shRNA targeting CA IX (shCAIX) incubated for 72 h. Twoindependent clones (C2, C5) expressing shCAIX were analyzed. Bottompanel, lysates were assessed by Western blot for CAIX expression.Beta-actin served as a loading control.

FIG. 5 includes representative images of TUNEL-positive cells (arrows)of (A) TOP 4T1 cells expressing shCAIX and cultured for 48 h in hypoxiaand the amount of cell death was compared to 4T1 cells expressing shNS.Top panel, representative images of TUNEL-positive cells (arrows). Scalebar=100 um. (A) Bottom panel, graph showing quantification of theTUNEL-positive cells by counting 5 random fields/cell line at 20×magnification. Data are expressed as fold change in TUNEL-positivecells, compared to control cells cultured in normoxia. n=5. (B) 4T1cells expressing shNS or shCAIX were cultured in the indicatedconditions for 72 hrs and intracellular levels of ATP were determined ontotal cell lysates. *P<0.01, compared to levels of ATP in normoxia.

FIG. 6 shows (A) representative bioluminescent images of spontaneousmetastasis using the 4T1 tumor model. Heat maps (light, least intense;darker, most intense;) are shown overlaid on gray-scale body images. (B)Same view, but with 4T1 cells expressing shNS or shCAIX and parental 4T1cells inoculated into the mammary gland of 10 BALB/c mice. * denotescompletion of primary tumor excision from the control groups.

FIG. 7 illustrates reduced primary tumor growth by human breast cancercells depleted of CAIX expression. MDA-MB-231 cells expressing shNS orshCAIX and parental MDA-MB-231 cells were subcutaneously inoculated intoflank of NOD.CB17-prkdc^(scid)/J mice and animals were monitored fortumor growth. n=7 for each group. Inset: MDA-MB-231 cells expressingshRNAmir targeting human CAIX (shCAIX) or a non-silencing controlsequence (shNS) were cultured in normoxia or hypoxia for 72 h andanalyzed for hypoxia-induced CAIX expression Western blot is shown.β-actin served as a loading control.

FIG. 8 shows that the depletion of CAIX inhibits of formation of lungmetastases in an experimental metastasis model. Panel (A) showsrepresentative bioluminescent images of experimental metastasis byCAIX-expressing 4T1 cells. Panel (B) shows the presence of metastaticnodules in gross images of mouse lungs from the 4T1 shNS group. Panel(C) shows quantification of the number of visible nodules in thedifferent tumor groups. Panel (D) shows immunohistochemical staining ofCAIX in 4TI cell-derived lung metastases.

FIG. 9 shows (A) the chemical structure of MST-017, or previouslyidentified “CAI-17” (Supuran, C. 2008, Nature. Vol 7: 168-181), (B)Cells were cultured for 72 h in the presence of 10 uM MST-017. Shown arerepresentative images of the FITC-tagged inhibitor bound to the celllines in the indicated conditions. (C) is a graphing of the change inextracellular pH for cells cultured for 72 h with or without MST-017(400, 600 and 400 uM for the 4T1, 66cl4 and 67NR cells, respectively).n=3. The mean changes in extracellular pH±s.e.m. are shown.

FIG. 10 shows data showing the in vivo efficacy of MST-017 to attenuatethe growth of 4T1 primary tumors. (A) 4T1 cells levels of CAIXexpression were analyzed by Western blot. (B) Left panel, tumor growthwas monitored by caliper-based measurement. Treatment initiation andtermination are indicated by arrows. Vehicle-treated and untreatedanimals served as controls. (C) The weights of treated animals weremonitored as a measure of general inhibitor toxicity. Mice were weighedjust prior to each dose of the CAIX inhibitor.

FIG. 11 shows the differences in 67NR tumor growth for animals treatedas graphed and by Western blot of CAIX. For (A), 67NR Cells werecultured in normoxia or hypoxia and levels of CAIX expression wereanalyzed by Western blot. For (B), animals were inoculated with 67NRcells and treated as for FIG. 10. The frame (C) graph shows data showingthat no significant differences in the weights among the varioustreatment and control arms were noted.

FIG. 12 shows (A) the chemical structure of CAIX inhibitor MST-119, and(B) representative bioluminescent images of metastases establishedfollowing intravenous injection 4T1 cells and treatment with MST-119.Frame (C) illustrates the results of quantification of tumor-derivedbioluminescence.

FIG. 13 shows (A) representative bioluminescent images of metastasesestablished following intravenous injection of 4T1 cells and treatmentwith MST-104. Frame (B) illustrates the results of quantification oftumor-derived bioluminescence.

FIG. 14 shows dose-dependent inhibition of growth of human breast tumorsimplanted orthotopically in the mammary gland and treated with CAIXinhibitor MST-104. Tumor growth was monitored over time. The inset panelshows that these cells up-regulate expression of CAIX when grown inhypoxia.

FIG. 15 shows that in contrast to the parental MDA-MB-231 human breastcancer cells, the highly lung-metastatic MDA-231 LM2-4 cells areinvasive when cultured in 3D Matrigel™ cultures in hypoxia.

FIG. 16 shows that ureido-sulfonamide inhibitors of CAIX (MST-104,MST-119, MST-107, MST-130) inhibit invasion of highly metastatic humanbreast cancer cells in 3D Matrigel™ cultures in hypoxia.

FIG. 17 shows differential effects ureido-sulfonamides on cell death in3D Matrigel™ cultures in hypoxia. (A) Representative images of thenumber of TUNEL-positive cells. Frame (B) illustrates the results ofquantification of TUNEL-positive cells.

FIG. 18 shows that genetic depletion of CAIX expression in 4T1 breastcancer cells (A) increases the number of cells required to initiatetumorsphere growth in hypoxia and (B) reduces the hypoxia-inducedincrease in CD44⁺/CD24^(−/low) cancer stem cells.

FIG. 19 shows that treatment of human breast cancer orthotopic tumorswith ureido-sulfonamide MST-104 depletes the cancer stem cell populationwithin the tumor. Frame (A) shows representative FACS plots sorting forESA+ cancer stem cells. Frame (B) illustrates the results ofquantification on the number of ESA+ human breast cancer stem cellspresent in the tumors.

DETAILED DESCRIPTION

Ureido-sulfonamides compositions suitable for the treatment ofmetastatic cancer are synthesized and utilized as herein described. Incertain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable excipient and a compound of Formula (I)

R-Q-Ar—SO₂NH₂

wherein R may be an aryl, heteroaryl, aralkyl alkyl or cycloalkyl group,any of the above groups may be further substituted, namely, they may bewith or without a substituent;

Q may be -L(CH₂)_(n)—, where n=0, 1 or 2 and L may be —NHC(X)NH—,—NHC(S)SNH—, —NHC(O)NHC(S)NH—, or —SO₂NH—, wherein X may be O or S; andAr may be a C₆-C₁₀ aryl or a heteroaryl group that contains at least oneheteroatom of oxygen, nitrogen or sulphur.

In certain embodiments, Q may be —NHC(O)NH—, Ar may be phenyl and R maybe PhCH₂, Ph₂CH, 4-FC₆H₄, 4-ClC₆H₄, 4-BrC₆H₄, C₆F₅, 2-MeOC₆H₄, 4-AcC₆H₄,2-i-PrC₆H₄, 4-i-PrC₆H₄, 4-n-BuC₆H₄, 4-n-BuOC₆H₄, 4-n-octyl-C₆H₄,4-NCC₆H₄, 2-NCC₆H₄, 4-PhOC₆H₄, 2-PhC₆H₄, 3-O₂NC₆H₄, 4-MeO-2-MeC₆H₃,Cyclopentyl, Indan-5-yl, 3,5-Me₂C₆H₃, 4-CF₃C₆H₄, or 3,5-(CF₃)₂C₆H₃.

Representative compounds include:

-   4-{[(Benzylamino)carbonyl]amino}benzenesulfonamide (MST-102);-   4-{[(Benzhydrylamino)carbonyl]amino}benzenesulfonamide (MST-103);-   4-{[(4′-Fluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-104);-   4-{[(4′-Bromophenyl)carbamoyl]amino}benzenesulfonamide (MST-105);-   4-{[(Pentafluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-107);-   4-{[(2′-Methoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-108);-   4-{[(4′-Acetylphenyl)carbamoyl]amino}benzenesulfonamide (MST-109);-   4-{[(2′-iso-Propylphenyl)carbamoyl]amino}benzenesulfonamide    (MST-110);-   4-{[(4′-iso-Propylphenyl)carbamoyl]amino}benzenesulfonamide    (MST-111);-   4-{[(4′-n-Butylphenyl)carbamoyl]amino}benzenesulfonamide (MST-112);-   4-{[(4′-Butoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-113);-   4-{[(4′-n-Octylphenyl)carbamoyl]amino}benzenesulfonamide (MST-114);-   4-{[(4′-Cyanophenyl)carbamoyl]amino}benzenesulfonamide (MST-115);-   4-{[(2′-Cyanophenyl)carbamoyl]amino}benzenesulfonamide (MST-116);-   4-{[(4′-Phenoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-117);-   4-{[(Biphenyl-2′-yl)carbamoyl]amino}benzenesulfonamide (MST-118);-   4-{[(3′-Nitrophenyl)carbamoyl]amino}benzenesulfonamide (MST-119);-   4-{[(4′-Methoxy-2′-methylphenyl)carbamoyl]amino}benzenesulfonamide    (MST-120);-   4-[(Cyclopentylcarbamoyl)amino]benzenesulfonamide (MST-122);-   4-{([(3′,5′-Dimethylphenyl)amino]carbonylamino)}benzenesulfonamide    (MST-123);-   4-{[(4′-Chlorophenyl)carbamoyl]amino}benzenesulfonamide (MST-124);-   4-{[(2′,3′-Dihydro-1H-inden-5′-ylamino]carbonylamino)}benzenesulfonamide    (MST-125);-   4-{[([4′-(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide    (MST-126);-   4-{[([3′,5′-bis(Trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide    (MST-127);-   3-(3-(4′-Iodophenyl)ureido)benzenesulfonamide (MST-128);-   3-(3-(4′-Fluorophenyl)ureido)benzenesulfonamide (MST-129);-   3-(3-(3′-Nitrophenyl)ureido)benzenesulfonamide (MST-130);-   3-(3-(4′-Acetylphenyl)ureido)benzenesulfonamide (MST-131);-   3-(3-(2′-Isopropylphenyl)ureido)benzenesulfonamide (MST-132);-   3-(3-(Perfluorophenyl)ureido)benzenesulfonamide (MST-133);-   4-(3-(4′-chloro-2-fluorophenyl)ureido)benzenesulfonamide (MST-134);-   4-(3-(4′-bromo-2′-fluorophenyl)ureido)benzenesulfonamide (MST-135);-   4-(3-(2′-fluoro-5′-nitrophenyl)ureido)benzenesulfonamide (MST-136);-   4-(3-(2′,4′,5′-trifluorophenyl)ureido)benzenesulfonamide (MST-137);-   4-(3-(2′-fluoro-5′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-138);-   4-(3-(2′-fluoro-3′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-139);-   4-(3-(2′,3′,4′-trifluorophenyl)ureido)benzenesulfonamide (MST-140);-   4-(3-(2′-fluorophenyl)ureido)benzenesulfonamide (MST-141);-   4-(3-(2′,4′-difluorophenyl)ureido)benzenesulfonamide (MST-142);-   4-(3-(3′-chlorophenyl)ureido)benzenesulfonamide (MST-143);-   4-(3-(2′,5′-dichlorophenyl)ureido)benzenesulfonamide (MST-144);-   4-(3-(2′-Chloro-5′-nitrophenyl)ureido)benzenesulfonamide (MST-145);-   4-(3-(2′-Chloro-4′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide    (MST-146);-   4-(3-(2′,6′-difluorophenyl)ureido)benzenesulfonamide (MST-147); or-   4-(3-(perchlorophenyl)ureido)benzenesulfonamide (MST-148).

As used herein, the C₆-C₁₀ aryl group means phenyl or 1, or 2-naphthyl,and a heteroaryl group means a C₂-C₁₂ heterocyclic aromatic compoundthat contains at least one heteroatom of oxygen, nitrogen or sulphur.Furthermore, 1,3,4-thiadiazole is a preferred heterocyclic group.

Further, an alkyl groups means a straight chain or branched, noncyclic,nonaromatic aliphatic hydrocarbon moiety. A cycloalkyl means a cyclichydrocarbon moiety. An aralkyl means an alkyl moiety substituted with anaryl. Examples of aralkyl includes benzyl (PhCH₂—) and diphenylmethyl(Ph₂CH—).

The term “substituted” in the context of alkyl, aryl, heteroaryl meansthat at least one hydrogen atom of the alky, aryl, and heteroaryl moietyis replaced with a substituent, i.e. a further chemical moiety. Suitablesubstituents include, for example, halogen (F, Cl, Br, I), cyano, alkyl,alkoxy (alkyl-O—), aryloxy (aryl-O—), nitro (—NO₂), cycloalkyl,haloalkyl (alkyl substituted by one or more halogens, e.g.,trifluoroalkyl), heteroaryl, and the like.

There is further provided compounds of formula (I), wherein thecompounds inhibits the activity of tumor-related CAIX and CAXII to agreater degree than it inhibits the activity of CAI and CAII as measuredin vitro.

A further embodiment provides a process for the preparation of thecompounds of formula (I). One preferred reaction is carried out betweena compound of formula R—NCX, wherein R and X are as defined above, and acompound of formula NH₂—Ar—SO₂NH₂ or NH₂CSNH—Ar—SO₂NH₂, wherein Ar asdefined above.

Another preferred reaction is the oxidative thiocarbamylation of acompound of formula NH₂—Y—SO₂NH₂ with sodium/potassiumN,N-dimethyl-/diethyldithiocarbamate, wherein Y is the group of—(CH₂)_(n)Ar—, n=0, 1, 2 and Ar is a C₆-C₁₀ aromatic or a heteroaromaticgroup that contains at least one heteroatom of oxygen, nitrogen orsulphur.

Reaction conditions are those known to those skilled in the art, see forexample, A. Scozzafava and C. T. Supuran Bioorg. Med. Chem. Lett. 2000,10, 1117-1120; C. T. Supuran; F. Briganti; S. Tilli; W. R. Chegwidden;A. Scozzafava Bioorg. Med. Chem. 2001, 9, 703-714; C. T. Supuran; A.Scozzafava; B. C. Jurca; M. A. Hies Eur. J. Med. Chem. 1998, 33, 83-93.

The following scheme shows an example of a process that may be used toprepare compounds according to the present disclosure.

The compounds according to the present disclosure are selective CAIX andCAXII inhibitors. Thus, there is further provided compounds of Formula(I), wherein the compounds inhibit the activity of tumor-related CAIXand CAXII to a greater degree than they inhibits the activity of CAI andCAII as measured in vitro. These compounds are useful in methods toreduce the growth of human breast cancer, to inhibit invasion by breastcancer cells under hypoxic conditions typical in solid tumors, to killhuman breast cancer cells in hypoxia, and to deplete cancer stem cellpopulations.

Cancer stem cells (CSCs) are defined as a subpopulation of cancer cellsthat have the properties of self renewal potential, the ability to giverise to non-CSC progeny, and greatly enhanced tumor-initiating potentialrelative to other cancer cells within the tumor (Chaffer and Weinberg,(2011) Science 331:1559-1564; Clevers, (2011) Nat. Med. 17: 313-319;Hanahan and Weinberg, (2011) Cell. 144: 646-674). CSCs are definedexperimentally as cells that have the ability to seed new tumors whenimplanted into an appropriate animal host (Chaffer and Weinberg, 2011;Hanahan and Weinberg, 2011). They are believed to be a component ofcancer therapy resistance.

TABLE 1 INHIBITION OF HCAI, HCAII (CYTOSOLIC ISOFORMS) AND HCAIX ANDHCAXII (TRANSMEMBRANE, TUMOR-ASSOCIATED ENZYMES) WITH UREIDOSULFONAMIDES. MST-101 TO MST-127.

K_(I) (nM) Activity Informal Name R hCAI hCAII hCAIX hCAXII MST-101 Ph760 3730 575 67.3 MST-102 PhCH₂ 92 2200 41.4 49.5 MST-103 Ph₂CH 83 372558.8 64.5 MST-104 4-FC₆H₄ 5080 9640 45.1 4.5 MST-105 4-BrC₆H₄ 1465 129069.3 7.9 MST-106 4-IC₆H₄ 5500 2634 24.5 4.3 MST-107 C₆F₅ 2395 5055 5.45.1 MST-108 2-MeOC₆H₄ 92 4070 465 61.2 MST-109 4-AcC₆H₄ 388 1060 5.4 4.6MST-110 2-i-PrC₆H₄ 9.0 3.3 0.5 4.2 MST-111 4-i-PrC₆H₄ 4330 5005 541 49.7MST-112 4-n-BuC₆H₄ 5530 2485 376 28.5 MST-113 4-n-BuOC₆H₄ 11.3 2.1 0.82.5 MST-114 4-n-octyl-C₆H₄ 536 9600 47.1 52.8 MST-115 4-NCC₆H₄ 57.0 64.76.0 6.5 MST-116 2-NCC₆H₄ 10.9 2.4 0.3 4.6 MST-117 4-PhOC₆H₄ 604 85 69.17.1 MST-118 2-PhC₆H₄ 1170 9.7 65.7 65.1 MST-119 3-O₂NC₆H₄ 23.4 15 0.95.7 MST-120 4-MeO-2-MeC₆H₃ 89.2 3310 73.3 6.0 MST-121 9H-fluoren-2-yl1700 908 102 55.4 MST-122 Cyclopentyl 470 2265 7.3 7.0 MST-1233,5-Me₂C₆H₃ 6530 1765 6.9 6.2 MST-124 4-ClC₆H₄ 2150 781 58 5.3 MST-125Indan-5-yl 9.8 8.9 7.0 2.5 MST-126 4-CF₃C₆H₄ 9.7 1150 6.2 2.3 MST-1273,5-(CF₃)₂C₆H₃ 3690 75 53 39

The compounds disclosed herein are useful for the preparation ofmedicaments as well as in a method for the treatment of a hypoxic tumorthat has CAIX or CAXII highly overexpressed. “Overexpression” means theexcessive expression of a gene, usually by producing too much of itseffect or product. The medicaments have inhibiting action toward CAIX,and are particularly effective for reversing acidification of a hypoxictumor and its surrounding environment.

The compounds disclosed herein are also capable of impairing and/oreradicating cancer stem cells. Cancer stem cells are thought to be abasis of resistance by tumors to traditional therapeutic agents ortechniques, such as chemotherapeutics or radiation.

In most cancer therapy, multiple agents with complementary modalities ofaction are typically used as part of a chemotherapy “cocktail.” It isanticipated that the compositions disclosed herein may be used in suchcocktail that may contain one or more additional antineoplastic agentsdepending on the nature of the cancer being treated. Otherchemotherapeutic agents, such as antimetabolites (i.e., 5-fluorouracil,floxuradine, thioguanine, cytarabine, fludarabine, 6-mercaptopurine,methotrexate, gemcitabine, capacitabine, pentostatin, trimetrexate, orcladribine); DNA crosslinking and alkylating agents (i.e., cisplatin,carboplatin, streptazoin, melphalan, chlorambucil, carmustine,methclorethamine, lomustine, bisulfan, thiotepa, ifofamide, orcyclophosphamide); hormonal agents (i.e., tamoxifen, roloxifen,toremifene, anastrozole, or letrozole); antibiotics (i.e., plicamycin,bleomycin, mitoxantrone, idarubicin, dactinomycin, mitomycin,doxorubicin or daunorubicin); immunomodulators (i.e., interferons, IL-2,or BCG); antimitotic agents (i.e., estramustine, paclitaxel, docetaxel,vinblastine, vincristine, or vinorelbine); topoisomerase inhibitors(i.e., topotecan, irinotecan, etoposide, or teniposide.); and otheragents (i.e., hydroxyurea, trastuzumab, altretamine, retuximab,L-asparaginase, or gemtuzumab ozogamicin) may therefor be used incombination with the compositions disclosed herein.

The molecules may be compounded with known pharmaceutical excipientssuch as salts, water, lipids, and/or simple sugars to arrive at aformulation suitable for injection, topical application, or ingestion.

Pharmaceutical formulation involves developing a preparation of thecompound which is both stable and acceptable for human use. Formulationsof the compounds will have been tested to ensure that the drug iscompatible with any solubilizing, stabilizing, lyophilizing, orhydrating agents.

The design of any formulation involves the characterization of a drug'sphysical, chemical, and mechanical properties in order to choose whatother ingredients should be used in the preparation.

Particle size, polymorphism, pH, and solubility, as all of these caninfluence bioavailability and hence the activity of a drug. The drugmust be combined with inactive additives by a method which ensures thatthe quantity of drug present is consistent in each dosage unit e.g. eachtablet.

It is unlikely that formulation studies will be complete by the timeclinical trials commence. This means that simple preparations aredeveloped initially for use in phase I clinical trials. Proof thelong-term stability of these formulations is not required, as they willbe used (tested) in a matter of days.

By the time phase III clinical trials are reached, the formulation ofthe drug should have been developed to be close to the preparation thatwill ultimately be used in the market. Stability studies are carried outto test whether temperature, humidity, oxidation, or photolysis(ultraviolet light or visible light) have any effect, and thepreparation is analyzed to see if any degradation products have beenformed.

In one embodiment, the compounds are formulated in polyethyleneglycolwith ethanol and saline. In one particular embodiment, the formulationconsists of 37.5% PEG400, 12.5% ethanol and 50% saline.

As used in this document, tumor may be taken to mean any primary ormetastatic cancer, hypoxic tumor tissue, or malignant growth. Any tumorsusceptible to hypoxia and/or metastases, particularly breast, lung,renal cancers, cervical, pancreatic, colorectal, glioblastoma, prostateand ovarian cancer may be treated according to embodiments disclosedherein. Methods are available to determine additional suitable cancertypes for treatment with the compositions disclosed herein, namely,methods are available and known to one of skill in the art for detectinghypoxic tissues. See, for example, U.S. Pat. Nos. 5,401,490 and5,843,404 which disclose methods of detecting hypoxia or hypoxictissues. Any of these techniques or others known to those skilled in theart may be used to identify hypoxic tissues.

Tumors susceptible to treatment will have elevated levels of CAIX orCAXII with respect to normal tissue. Isozymes CAIX and CAXII arepredominantly found in tumor cells and show a restricted expression innormal tissues. It has been recently proven that by efficientlyhydrating carbon dioxide to protons and bicarbonate, these CAscontribute significantly to the extracellular acidification of solidtumors, whereas their inhibition reverses this phenomenon to a certainextent. CAIX and CAXII are overexpressed in many such tumors in responseto the hypoxia inducible factor (HIF) pathway.

As demonstrated in the data, CAIX and CAXII are associated with hypoxiaand metastases. Thus a hypoxic and metastatic tumor would not need to betested to prove elevated levels of CAIX and CAXII to indicate treatmentusing the compounds disclosed herein because of the data alreadysupporting the supposition. However, U.S. Pat. No. 7,378,091 by Gudas etal. discloses CAIX antibodies useful in detection and diagnosis.Antibodies against CAXII, as well as RNA probes, can be used to assessoverexpression of CAXII in biopsied tumor samples.

CA XII is also assessed in Battke et al., (2011) Cancer ImmunolImmunother. May; 60(5):649-58.

Tumor growth, persistence and/or spread may be said to be suppressed bycompounds disclosed herein, or by their use in treating mammals soafflicted. “Suppression” in this application may mean induction ofregression, inhibition of growth, and inhibition of spread, especiallyas these terms relate to tumors and cancers suffered by mammals,particularly humans.

Typical chemotherapeutic agents including, but not limited to docetaxel,vinca alkaloids, mitoxanthrone, cisplatin, paclitaxel, 5-FU, Herceptin,Avastin, Gleevec may be used in combination with the compounds disclosedherein. Similarly, radiation therapy may be combined with administrationschedules including the compounds disclosed herein.

When surgical intervention is performed, the compounds and compositionsdisclosed herein may be used preoperatively, perioperatively, orpost-operatively. Dosage is typically determined by dosing schemes whichuse patient size and weight to calculate the patient's body surfacearea, which correlates with blood volume, to determine initial dosing.Starting dosages are generally worked out during clinical testing oftherapeutic compounds.

The background and current approaches for the clinical approach to tumortreatment may be found in Takimoto C H, Calvo E. “Principles ofOncologic Pharmacotherapy” in Pazdur R, Wagman L D, Camphausen K A,Hoskins W J (Eds) Cancer Management: A Multidisciplinary Approach. 11ed. 2008, which is freely available athttp://www.cancernetwork.com/cancer-management-11/chapter03/article/10165/1402628.

The following examples are used to illustrate aspects disclosed herein,but the embodiments are not intended to be limited by theseillustrations.

EXAMPLES Example 1 Preparations of Specific Embodiments (CompoundsMST-101 to MST-127 Inclusive) General Procedure for the Preparation ofCompounds of Formula (I)

Methods in chemistry: ¹H, ¹³C and ¹⁹F spectra were recorded using aBruker Advance 111400 MHz spectrometer. The chemical shifts are reportedin parts per million (ppm) and the coupling constants (J) are expressedin Hertz (Hz). Infrared spectra were recorded on a Perkin Elmer SpectrumR XI spectrometer as solids on KBr plates. Melting points (m.p.) weremeasured in open capillary tubes, unless otherwise stated, using a BuchiMelting Point B-540 melting point apparatus, and are uncorrected. Thinlayer chromatography (TLC) was carried out on Merck silica gel 60 F₂₅₄aluminum backed plates. Elution of the plates was carried out usingethyl acetate-petroleum ether as eluting system. Visualization wasachieved with UV light at 254 nm, by dipping into a ninhydrin TLC stainsolution and heating with a hot air gun. Flash column chromatography wascarried out using silica gel (obtained from Aldrich Chemical Co., Milan,Italy) as the adsorbent. The crude product was introduced into thecolumn as a solution in the same elution solvent system. Solvents andchemicals were used as supplied from Aldrich Chemical Co., Milan, Italy.

4-Aminobenzenesulfonamide (2.9 mmole) was dissolved in acetonitrile(20-30 mL) and then treated with a stoichiometric amount of anisocyanide. The mixture was stirred at r.t. or heated at 50° C. for 2hours, until completion (TLC monitoring). The heavy precipitate formedwas filtered-off, washed with diethyl ether and dried under vacuum.

4-[(anilinocarbonyl)amino]benzenesulfonamide (MST-101)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with phenylisocyanate (0.23 g; 2.90 mmols) and the reaction was stirred at r.t. for1 day, treated as described in the general procedure previously reportedto give MST-101 as a white solid in 43.7% yield. m. p. 233-235° C.(Lie); silica gel TLC R_(f) 0.63 (ethyl acetate/petroleum ether 33%);v_(max) (KBr) cm⁻¹, 3340 (N—H urea), 1656 (C═O urea), 1595 (aromatic);δ_(H) (400 MHz, DMSO-d₆) 7.00 (1H, tt, J 7.4, 0.8, 4′-H), 7.20 (2H, s,SO₂NH₂), 7.29 (2H, dd, J 8.2, 0.8, 2×3′-H), 7.47 (2H, dd, J 8.2, 1.2,2×2′-H), 7.61 (2H, d, J 9.0, 2×3-H), 7.73 (2H, d, J 9.0, 2×2-H), 8.82(1H, s, NH), 9.09 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═Ourea), 143.8, 140.2, 137.8, 129.8, 127.7, 123.1, 119.4, 118.4.

4-{[(Benzylamino)carbonyl]amino}benzenesulfonamide (MST-102)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with benzylisocyanate (0.39 g; 2.90 mmols) and the reaction was stirred at r.t. for4 h, treated as described in the general procedure previously reportedto give MST-102 as a white solid in 42.3% yield. m.p. 194-196° C.;silica gel TLC R_(f) 0.58 (ethyl acetate/petroleum ether 33%); v_(max)(KBr) cm⁻¹, 3313 (N—H urea), 1674 (C═O urea), 1591 (aromatic); δ_(H)(400 MHz, DMSO-d₆) 4.35 (2H, d, J 6.0, 1′-H₂), 6.81 (1H, t, J 6.0, NH),7.19 (2H, s, SO₂NH₂), 7.28 (1H, tt, J 6.8 2.0, 5′-H), 7.35 (4H, m,2×3′-H, 2×4′-H), 7.59 (2H, d, J 9.0, 2×3-H), 7.71 (2H, d, J 9.0, 2×2-H),9.0 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 155.8 (C═O urea), 144.5,141.0, 137.1, 129.3, 128.1, 127.8, 127.7, 117.8, 43.7 (0-1′).

4-{[(Benzhydrylamino)carbonyl]amino}benzenesulfonamide (MST-103)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated withbenzhydryl isocyanate (0.61 g; 2.90 mmols) and the reaction was stirredfor 2 h, treated as described in the general procedure previouslyreported to give MST-103 as a white solid in 42.4% yield. m.p. 235-236°C.; silica gel TLC R_(f) 0.76 (ethyl acetate/petroleum ether 33%);v_(max) (KBr) cm⁻¹, 3338 (N—H urea), 1696 (C═O urea), 1592 (aromatic);δ_(H) (400 MHz, DMSO-d₆) 6.0 (1H, d, J 7.6, NH), 7.19 (2H, s, SO₂NH₂),7.29 (2H, tt, J 7.2 1.6, 2×4′-H), 7.38 (9H, m, 1′-H, 4×3′-H, 4×4′-H)7.56 (2H, d, J 8.8, 2×3-H), 7.70 (2H, d, J 8.8, 2×2-H), 8.9 (1H, s, NH);δ_(C) (100 MHz, DMSO-d₆) 154.9 (C═O urea), 144.2, 143.8, 137.2, 129.5,127.9, 127.8, 127.7, 117.7, 57.8 (C-1′).

4-{[(4′-Fluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-104)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-fluorophenyl isocyanate (0.40 g; 2.90 mmols) and the reaction wasstirred at r.t. for 2 days, treated as described in the generalprocedure previously reported to give MST-104 as a white solid in 55.5%yield. m.p. 242-243° C.; silica gel TLC R_(f) 0.53 (ethylacetate/petroleum ether 33%); v_(max) (KBr) cm⁻¹, 3338 (N—H urea), 1697(C═O urea), 1593 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.17 (2H, t, J9.0, 2×2′-H), 7.24 (2H, s, SO₂NH₂), 7.51 (2H, dd, J 9.0 4.8, 2×3′-H),7.64 (2H, d, J 8.8, 2×3-H), 7.76 (2H, d, J 8.8, 2×2-H), 8.86 (1H, s,NH), 9.09 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 158.5 (d, J_(C-F) 237,C-4′), 153.3 (C═O urea), 143.7, 137.8, 136.6 (d, ⁴J_(C-F) 3, C-1′),127.7, 121.2 (d, ³J_(C-F) 7, C-2′), 118.4, 116.3 (d, ²J_(C-F) 22, C-3′);δ_(F) (376.5 MHz, DMSO-d₆) −121.0 (1F, s).

4-({[(4′-Bromophenyl)amino]carbonyl}amino)benzenesulfonamide (MST-105)

4-Amino-benzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-bromophenyl isocyanate (0.57 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-105 as a white solid in 43.1% yield.m.p. 269-271° C.; silica gel TLC R_(f) 0.38 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3328 (N—H urea), 1652 (C═O urea), 1590(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.24 (2H, s, SO₂NH₂), 7.49 (2H, d,J 9.2, 2×2′-H), 7.51 (2H, d, J 9.2, 2×3′-H), 7.64 (2H, d, J 8.8, 2×3-H),7.76 (2H, d, J 8.8, 2×2-H), 8.99 (1H, s, NH), 9.15 (1H, s, NH); δ_(C)(100 MHz, DMSO-d₆) 152.6 (C═O urea), 143.1, 139.3, 137.5, 132.0, 127.3,120.9, 118.1, 114.1.

4-{[(4′-Iodophenyl)carbamoyl]amino}benzenesulfonamide (MST-106)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-iodophenyl isocyanate (0.71 g; 2.90 mmols) and the reaction wasstirred at r.t. overnight, treated as described in the general procedurepreviously reported to give MST-106 as a white solid in 46.5% yield.m.p. 275-277°; silica gel TLC R_(f) 0.55 (ethyl acetate/petroleum ether33%) v_(max) (KBr) cm⁻¹, 3325 (N—H urea), 1652 (C═O urea), 1586(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.24 (2H, s, SO₂NH₂), 7.36 (2H, d,J 8.8, 2×2′-H), 7.63 (2H, d, J 6.8, 2×3-H), 7.66 (2H, d, J 6.8, 2×2-H),7.77 (2H, d, J 8.8, 2×3′-H), 8.97 (1H, s, NH), 9.15 (1H, s, NH); δ_(C)(100 MHz, DMSO-d₆), 153.2 (C═O urea), 143.7, 140.3, 138.5, 138.1, 127.9,121.7, 118.6, 86.2 (C-4′).

4-{[(Pentafluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-107)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated withpentafluorophenyl isocyanate (0.60 g; 2.90 mmols) and the reaction wasstirred r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-107 as a white solid in 97.7% yield.m.p. 251-253° C.; silica gel TLC R_(f) 0.49 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3329 (N—H urea), 1656 (C═O urea), 1597(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.23 (2H, s, SO₂NH₂), 7.61 (2H, d,J 8.8, 2×3-H), 7.74 (2H, d, J 8.8, 2×2-H), 8.65 (1H, s, NH), 9.48 (1H,s, NH); δ_(C) (100 MHz, DMSO-d₆) 152.9 (C═O urea), 144.0 (m, J_(C-F)239, C-2′), 143.3, 139.6 (m, J_(C-F) 248, C-4′), 138.5, 138.2 (m,J_(C-F) 249, C-3′), 127.8, 118.8, 114.7 (ddd, ²J_(C-F 23,) ³J_(C-F) 14,⁴J_(C-F) 4, C-1′); δ_(F) (376.5 MHz, DMSO-d₆) −146.2 (2F, dd, ³J 24, ⁴J5.1, 2×2′-F), −159.2 (2F, t, ³J 23, 2×4′-F), −164.0 (1 F, dd, ³J 23.3,⁴J 5.0, 2×3′-F).

4-{[(2′-Methoxyphenyl)amino]carbonyl)}aminobenzenesulfonamide (MST-108)

4-Amino-benzenesulfanilamide (0.50 g; 2.90 mmols) was treated with2-methoxyphenyl isocyanate (0.43 g; 2.90 mmols) and the reaction wasstirred at r.t. overnight, treated as described in the general procedurepreviously reported to give MST-108 as a white solid in 40.4% yield.m.p. 234-236° C.; silica gel TLC R_(f) 0.47 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3362 (N—H urea), 2838 (C—H aliphatic),1684 (C═O urea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 3.92 (3H, s,CH₃), 6.94 (1H, ddd, J 8.2 7.4 1.4, 4′-H), 7.01 (1H, ddd, J 8.0 7.4 1.6,5′-H), 7.07 (1H, dd, J 8.2 1.2, 3′-H), 7.23 (2H, s, SO₂NH₂), 7.64 (2H,d, J 8.8, 2×2-H), 7.77 (2H, d, J 8.8, 2×3-H), 8.16 (1H, dd, J 8.0 1.6,6′-H), 8.38 (1H, s, NH), 9.73 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆)153.0 (C═O, urea), 148.7, 143.8, 137.7, 129.2, 127.8, 123.2, 121.5,119.4, 118.1, 111.7, 56.7 (CH₃).

4-{([(4′-Acetylphenyl)amino]carbonyl)amino}benzenesulfonamide (MST-109)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-acetylphenyl isocyanate (0.46 g; 2.90 mmols) and the reaction wasstirred at r.t for 1 day, treated as described in the general procedurepreviously reported to give MST-109 as a white solid in 46.6% yield.m.p. 258-260° C.; silica gel TLC R_(f) 0.27 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3300 (N—H urea), 1659 (C═O urea), 1590(aromatic); δ_(H) (400 MHz, DMSO-d₆) 2.56 (3H, s, CH₃), 7.26 (2H, s,SO₂NH₂), 7.64 (2H, d, J 8.8, 2×2′-H), 7.67 (2H, d, J 8.8, 2×3-H), 7.79(2H, d, J 8.8, 2×2-H), 7.96 (2H, d, J 8.8, 2×3′-H), 9.22 (1H, s, NH),9.25 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 197.3 (C═O), 152.9 (C═Ourea), 144.9, 143.4, 138.2, 131.7, 130.6, 127.8, 118.7, 118.4, 27.3(CH₃).

4-{([(2′-Isopropylphenyl)amino]carbonyl)amino}benzenesulfonamide(MST-110)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with2-isopropylphenyl isocyanate (0.47 g; 2.90 mmols) and the reaction wasstirred at r.t. for 6 h, treated as described in the general procedurepreviously reported to give MST-110 as a white solid in 48.7% yield.m.p. 226-227° C.; silica gel TLC R_(f) 0.65 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3361 (N—H urea), 2966 (C—H aliphatic),1676 (C═O urea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 1.24 (6H, d,J 6.8, 2×CH₃), 3.19 (1H, sept, J 6.8, CH), 7.14 (1H, ddd, J 7.9 7.6 1.6,4′-H), 7.19 (ddd, J 7.9 6.8 1.2, 5′-H), 7.23 (2H, s, SO₂NH₂), 7.34 (1H,dd, J 7.6 1.6, 3′-H), 7.65 (2H, d, J 8.8, 2×3-H), 7.68 (1H, dd, J 6.81.2, 6′-H), 7.76 (2H, d, J 8.8, 2×2-H), 8.11 (1H, s, NH), 9.37 (1H, s,NH); δ_(C) (100 MHz, DMSO-d₆) 153.8 (C═O urea), 144.0, 140.8, 137.6,136.0, 127.8, 126.7, 126.3, 125.3, 124.8, 118.2, 27.8 (CH), 24.1(2×CH₃).

4-{([(4′-Isopropylphenyl)amino]carbonyl)amino}benzenesulfonamide(MST-111)

4-Amino-benzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-isopropylphenyl isocyanate (0.47 g; 2.90 mmols) and the reaction wasstirred at r.t. for 3 h, treated as described in the general procedurepreviously reported to give MST-111 as a white solid in 58.5% yield.m.p. 226-227° C.; silica gel TLC R_(f) 0.50 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3351 (N—H urea), 2964 (C—H aliphatic),1647 (C═O urea), 1590 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 1.22 (6H, d,J 6.8, 2×CH₃), 2.87 (1H, sept, J 6.8, CH), 7.20 (2H, d, J 8.4, 2×3′-H),7.23 (2H, s, SO₂NH₂), 7.40 (2H, d, J 8.4, 2×2′-H), 7.64 (2H, d, J 9.0,2×3-H), 7.76 (2H, d, J 9.0, 2×2-H), 8.72 (1H, s, NH), 9.04 (1H, s, NH);δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═O urea), 143.9, 143.2, 137.9, 137.6,127.7, 127.5, 119.5, 118.3, 33.7 (CH), 24.9 (CH₃).

4-({[(4′-Butylphenyl)amino]carbonyl}amino)benzenesulfonamide (MST-112)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-butylphenyl isocyanate (0.51 g; 2.90 mmols) and the reaction wasstirred at r.t. for 3 h, treated as described in the general procedurepreviously reported to give MST-112 as a white solid in 48.6% yield.m.p. 243-245° C.; silica gel TLC R_(f) 0.54 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3333 (N—H urea), 2929 (C—H aliphatic),1653 (C═O urea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 0.93 (3H, t,J 7.2, CH₃), 1.33 (2H, six, J 7.4, CH₂), 1.57 (2H, sept, J 7.4, CH₂),2.56 (2H, t, J 7.6, CH₂), 7.14 (2H, d, J 8.6, 2×3′-H), 7.23 (2H, s,SO₂NH₂), 7.39 (2H, d, J 8.6, 2×2′-H), 7.63 (2H, d, J 9.0, 2×3-H), 7.76(2H, d, J 9.0, 2×2-H), 8.71 (1H, s, NH), 9.04 (1H, s, NH); δ_(C) (100MHz, DMSO-d₆) 153.2 (C═O urea), 143.9, 137.8, 137.6, 137.1, 129.5,127.3, 119.5, 118.3, 35.1 (CH₂), 34.2 (CH₂), 22.6 (CH₂), 14.7 (CH₃).

4-{[(4′-Butoxyphenyl)carbamoyl]amino}benzenesulfonamide (MST-113)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-butoxyphenyl isocyanate (0.55 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-113 as a white solid in 46.0% yield.m.p. 236-239° C.; silica gel TLC R_(f) 0.60 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3361 (N—H urea), 2957 (C—H aliphatic),1646 (C═O urea), 1592 (aromatic); δ_(H) (400 MHz DMSO-d₆) 0.97 (3H, t, J7.6, CH₃), 1.48 (2H, six, J 7.2, CH₂), 1.71 (2H, six, J 6.8, CH₂), 3.96(2H, t, J 6.4, CH₂), 6.90 (2H, d, J 9.2, 2×3′-H), 7.22 (2H, s, SO₂NH₂),7.38 (2H, d, J 9.2, 2×2′-H), 7.63 (2H, d, J 8.8, 2×3-H), 7.75 (2H, d, J8.8, 2×2-H), 8.62 (1H, s, NH), 9.02 (1H, s, NH); δ_(C) (100 MHz,DMSO-d₆) 155.1 (C═O urea), 153.3, 144.0, 137.5, 133.1, 127.7, 121.2,118.2, 115.5, 68.2 (OCH₂), 31.7 (CH₂), 19.7 (CH₂), 14.6 (CH₃).

4-{([(4-Octylphenyl)amino]carbonyl)amino}benzenesulfonamide (MST-114)

4-Aminobenzenesulphanilamide (0.50 g; 2.90 mmols) was treated with4-octylphenyl isocyanate (0.67 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-114 as a white solid in 41.4% yield.m.p. 282-283° C.; silica gel TLC Rf 0.59 (ethyl acetate/petroleum ether33%); v_(max) (KBr) cm⁻¹, 3332 (N—H urea), 2924 (C—H aliphatic), 1653(C═O urea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 0.89 (3H, t, J6.8, CH₃), 1.30 (12H, m, CH₂), 1.57 (2H, t, J 7.6, CH₂), 7.14 (2H, d, J8.6, 2×3′-H), 7.23 (2H, s, SO₂NH₂), 7.39 (2H, d, J 8.6, 2×2′-H), 7.63(2H, d, J 9.0, 2×3-H), 7.76 (2H, d, J 9.0, 2×2-H), 8.72 (1H, s, NH),9.05 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆), 153.2 (C═O urea), 143.9,137.8, 137.6, 137.1, 129.5, 127.7, 119.5, 118.2, 35.4 (CH₂), 32.2 (CH₂),32.0 (CH₂), 29.7 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 23.0 (CH₂), 14.9 (CH₃).

4-{[(4′-Cyanophenyl)carbamoyl]amino}benzenesulfonamide (MST-115)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated4-cyanophenyl isocyanate (0.42 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-115 as a white solid in 60.6% yield m.p.265-267° C.; silica gel TLC R_(f) 0.37 (ethyl acetate/petroleum ether33%); v_(max) (KBr) cm⁻¹, 3355 (N—H urea), 2221 (C≡N), 1695 (C═O urea),1594 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.26 (2H, s, SO₂NH₂), 7.66(2H, d, J 8.8), 7.68 (2H, d, J 9.2), 7.78 (2H, d, J 8.8, overlapping),7.79 (2H, d, J 8.8, overlapping), 9.28 (1H, s, NH), 9.35 (1H, s, NH);δ_(C) (100 MHz, DMSO-d₆) 152.8 (C═O urea), 144.8, 143.2, 138.3, 134.2,127.8, 120.1, 119.2, 118.8, 104.6 (C≡N).

4-{([(2′-Cyanophenyl)amino]carbonyl)amino}benzenesulfonamide (MST-116)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with2-cyanophenyl isocyanate (0.41 g; 2.90 mmols) and the reaction wasstirred for 1 day until a precipitate was formed. The crude obtained waspurified by silica gel column chromatography eluting with ethylacetate/petroleum ether 1/1 to give MST-116 as a white solid. m.p.256-258° C.; silica gel TLC R_(f) 0.73 (ethyl acetate/petroleum ether33%); v_(max) (KBr) cm⁻¹, 3310 (N—H urea), 2231 (C≡N), 1696 (C═O urea),1587 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.26 (1H, ddd, J 7.6 7.2 0.8,4′-H), 7.27 (2H, s, SO₂NH₂), 7.67 (2H, d, J 9.0, 2×3-H), 7.70 (1H, ddd,J 8.4 7.2 1.6, 5′-H), 7.80 (2H, d, J 9.0, 2×2-H), 7.82 (1H, dd, 7.6 1.6,3′-H), 8.1 (1H, dd, J 8.4 0.4, 6′-H), 8.90 (1H, s, NH), 9.78 (1H, s,NH); δ_(C) (100 MHz, DMSO-d₆) 152.8 (C═O urea), 143.2, 142.4, 138.4,135.0, 134.1, 127.8, 124.5, 122.6, 118.7, 117.8, 103.6 (C≡N).

4-({[(4′-Phenoxyphenyl)amino]carbonyl}amino)benzenesulfonamide (MST-117)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with 4phenoxyphenyl isocyanate (0.61 g; 2.90 mmols) and the reaction wasstirred at r.t. for 5 h, treated as described in the general procedurepreviously reported to give MST-117 as a white solid in 46.8% yield.m.p. 236-237° C.; silica gel TLC R_(f) 0.41 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3328 (N—H urea), 1653 (C═O urea), 1595(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.00 (2H, dd, J 8.8 1.2, 3′-H),7.03 (2H, d, J 9.0, 6′-H), 7.13 (1H, dt, J 7.5 0.8, 8′-H), 7.24 (2H, s,SO₂NH₂), 7.40 (2H, dd, J 9.0 7.5, 7′-H), 7.52 (2H, d, J 8.8, 2′-H), 7.65(2H, d, J 9.2, 3-H), 7.76 (2H, d, J 9.2, 2-H), 8.84 (1H, s, NH), 9.08(1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 158.5 (C═O urea), 153.3, 151.9,143.8, 137.7, 136.2, 130.9, 127.7, 123.8, 121.2, 120.7, 118.7, 118.4.

4-[(Biphenyl-2′-ylcarbamoyl)amino]benzenesulfonamide (MST-118)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated withbiphenyl-2-yl isocyanate (0.57 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-118 as a white solid in 40.0% yield.m.p. 229-231° C.; silica gel TLC R_(f) 0.75 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3365 (N—H urea), 1675 (C═O urea), 1584(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.20 (1H, d, J 7.4), 7.22 (2H, s,SO₂NH₂), 7.27 (1H, dd, J 7.4 1.6), 7.39 (1H, dt, J 8.4 1.6, 10′-H), 7.46(2H, dt, J 6.8 1.6), 7.54 (1H, d, J 7.6, 6′-H), 7.58 (2H, d, J 9.2,2×3-H), 7.74 (2H, d, J 9.2, 2×2-H), 7.83 (1H, s, NH), 7.95 (1H, d, J8.0, 3′-H), 9.41 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 153.6 (C═O urea),144.0, 139.5, 137.8, 136.4, 134.2, 131.5, 130.2, 129.9, 128.9, 128.6,127.9, 124.8, 124.0, 118.3.

4-{[(3′-Nitrophenyl)carbamoyl]amino}benzenesulfonamide (MST-119)

4-Aminobenzenesulfanilamide (0.50 mg; 2.90 mmols) was treated with3-nitrophenyl isocyanate (0.47 g; 2.90 mmols) and the reaction wasstirred at r.t. for 1 day, treated as described in the general procedurepreviously reported to give MST-119 as a yellow solid in 44.3% yield.m.p. 246-248° C.; silica gel TLC R_(f) 0.39 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3370 (N—H urea), 1709 (C═O urea), 1592(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.23 (2H, s, SO₂NH₂), 7.59 (1H, dd,J 8.4 8.0, 5′-H), 7.65 (2H, d, J 9.0, 2×3-H), 7.73 (1H, ddd, J 8.4 2.0,0.8, 6′-H), 7.76 (2H, d, J 9.0, 2×2-H), 7.86 (1H, ddd, J 8.0 2.4 0.8,4′-H), 8.58 (1H, appt, J 2.2, 2′-H), 9.25 (1H, s, NH), 9.35 (1H, s, NH);δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═O urea), 149.1, 143.3, 141.6, 138.3,131.1, 127.7, 125.5, 118.8, 117.6, 113.3.

4-{([(4′-Methoxy-2′-methylphenyl)amino]carbonyl)amino}benzenesulfonamide(MST-120)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-methoxy-2-methylphenyl isocyanate (0.47 g; 2.90 mmols) and thereaction was stirred at r.t. overnight, treated as described in thegeneral procedure previously reported to give MST-120 as a white solidin 40.1% yield. m.p. 240-241° C.; silica gel TLC R_(f) 0.35 (ethylacetate/petroleum ether 33%); v_(max) (KBr) cm⁻¹, 3313 (N—H urea), 2835(C—H aliphatic), 1647 (C═O urea), 1591 (aromatic); δ_(H) (400 MHz,DMSO-d₆) 2.25 (3H, s, CH₃), 3.76 (3H, s, OCH₃), 6.78 (1H, dd, J 8.8 2.8,5′-H), 6.84 (1H, d, J 2.8, 3′-H), 7.22 (2H, s, SO₂NH₂), 7.55 (1H, d, J8.8, 6′-H), 7.63 (2H, d, J 8.8, 2×3-H), 7.75 (2H, d, J 8.8, 2×2-H), 7.96(1H, s, NH), 9.26 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 156.6 (C═Ourea), 153.7, 144.2, 137.4, 132.3, 130.7, 127.8, 125.3, 118.1, 116.4,112.2, 56.1 (OCH₃), 19.0 (CH₃).

4-{[(9H-Fluoren-2-ylamino)carbonyl]amino}benzenesulfonamide (MST-121)

To a solution of 4-aminobenzenesulfanilamide (0.50 g; 2.90 mmols) inacetonitrile (20 ml) was added dropwise 9H-fluoren-2-yl isocyanate (0.59g; 2.90 mmols) dissolved in 10 ml of acetonitrile. The reaction wasstirred for 1 h at r.t., treated as described in the general procedurepreviously reported to give MST-121 as a white solid in 62.0% yield.m.p. 280-285° C.; silica gel TLC R_(f) 0.52 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3329 (N—H urea), 1648 (C═O urea), 1591(aromatic); δ_(H) (400 MHz, DMSO-d₆) 3.94 (2H, s, CH₂), 7.24 (2H, s,SO₂NH₂), 7.29 (1H, appt, J 7.2, 4′-H), 7.39 (1H, appt, J 7.2, 5′-H),7.46 (1H, d, J 7.2, 3′-H), 7.58 (1H, d, J 7.2, 6′-H), 7.67 (1H, d, J8.4, 2×3-H), 7.78 (1H, d, J 8.4, 2×2-H), 7.83 (3H, m, 2′-H, 7′-H, 8′-H),8.94 (1H, s, NH), 9.14 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═Ourea), 144.9, 143.8, 143.5, 142.0, 139.3, 137.7, 136.4, 127.8, 127.6,126.8, 125.9, 121.2, 120.2, 118.4, 118.2, 116.2, 37.4 (CH₂).

4-[(Cyclopentylcarbamoyl)amino]benzenesulfonamide (MST-122)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated withcyclopentyl isocyanate (0.32 g; 2.90 mmols) and the reaction was stirredat 50° C. for 2 h, treated as described in the general procedurepreviously reported to give MST-122 as a white solid in 68.8% yield.m.p. 224-226° C.; silica gel TLC R_(f) 0.57 (ethyl acetate/petroleumether 33%); v_(max) (KBr) cm⁻¹, 3328 (N—H urea), 3055 (C—H aliphatic),1684 (C═O urea), 1591 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 1.41 (2H, m,2×3′-H_(ax)), 1.58 (2H, m, 2×2′-H_(ax)), 1.67 (2H, m, 2×2′-H_(eq)), 1.88(2H, m, 2×3′-H_(eq)), 3.98 (1H, six, J 6.8, 1′-H), 6.35 (1H, d, J 6.8,NH), 7.18 (2H, s, SO₂NH₂), 7.55 (2H, d, J 8.8, 2×3-H), 7.70 (2H, d, J8.8, 2×2-H), 8.68 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 155.3 (C═Ourea), 144.6, 136.8, 127.7, 117.6, 51.8 (C-1′), 33.7 (C-2′), 24.1(C-3′).

4-{([(3,5-dimethylphenyl)amino]carbonylamino)}benzenesulfonamide(MST-123)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with3,5-dimethylphenyl isocyanate (0.43 g; 2.90 mmols) and the reaction wasstirred at r.t. overnight, treated as described in the general procedurepreviously reported to give MST-123 as a white solid in 60.6% yield. m.p235-236° C.; silica gel TLC R_(f) 0.58 (ethyl acetate/petroleum ether33%); v_(max) (KBr) cm⁻¹, 3343 (N—H urea), 2860 (C—H aliphatic), 1686(C═O urea), 1595 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 2.27 (6H, s,2×CH₃), 6.68 (1H, s, 4′-H), 7.12 (2H, s, 2′-H), 7.23 (2H, s, SO₂NH₂),7.64 (2H, d, J 8.8, 2×3-H), 7.76 (2H, d, J 8.8, 2×2-H), 8.67 (1H, s,NH), 9.06 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 153.1 (C═O, urea),143.8, 140.1, 138.7, 137.7, 127.7, 124.7, 118.3, 117.1, 22.0 (2×CH₃).

4-{([(4′-chlorophenyl)amino]carbonylamino)}benzenesulfonamide (MST-124)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-chlorophenyl isocyanate (0.44 g; 2.90 mmols) and the reaction wasstirred at r.t. overnight, treated as described in the general procedurepreviously reported to give MST-124 as a white solid in 89.4% yield. m.p239-240° C.; silica gel TLC R_(f) 0.44 (ethyl acetate/petroleum ether33%); v_(max) (KBr) cm⁻¹, 3327 (N—H urea), 1652 (C═O urea), 1592(aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.24 (2H, s, SO₂NH₂), 7.38 (2H, d,J 8.8, 2×2′-H), 7.53 (2H, d, J 8.8, 2×3′-H), 7.64 (2H, d, J 8.8, 2×3-H),7.76 (2H, d, J 8.8, 2×2-H), 8.97 (1H, s, NH), 9.13 (1H, s, NH); δ_(C)(100 MHz, DMSO-d₆) 153.1 (C═O, urea), 143.6, 139.2, 137.9, 129.6, 127.7,126.6, 120.9, 118.5.

4-{[(2,3-dihydro-1H-inden-5-ylamino]carbonylamino)}benzenesulfonamide(MST-125)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with5-indanylisocyanate (0.46 g; 2.90 mmols) and the reaction was stirred atr.t. overnight, treated as described in the general procedure previouslyreported to give MST-125 as a white solid in 60.6% yield. m.p 233-235°C.; silica gel TLC R_(f) 0.61 (ethyl acetate/petroleum ether 33%);v_(max) (KBr) cm⁻¹, 3333 (N—H urea), 2844 (C—H aliphatic), 1653 (C═Ourea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 2.04 (2H, appqt, J 7.4,2′-H), 2.85 (4H, appqt, J 7.4, 2×1′-H, 2×3′-H), 7.16 (1H, appd, J 8.2,6′-H), 7.19 (1H, appdd, J 8.2 1.6, 7′-H), 7.25 (2H, s, SO₂NH₂), 7.42(1H, s, 4′-H), 7.63 (2H, d, J 8.8, 2×3-H), 7.75 (2H, d, J 8.8, 2×2-H),8.70 (1H, s, NH), 9.06 (1H, s, NH); δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═O,urea), 145.2, 143.9, 138.4, 137.6, 127.8, 125.2, 118.3, 117.7, 115.7,one carbon overlapping signal, 33.48, 32.64, 26.15 (3×CH₂).

4-{[([4-(trifluoromethyl)phenyl]amino}carbonyl)amino]benzenesulfonamide(MST-126)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with4-trifluoromethyl-phenylisocyanate (0.54 g; 2.90 mmols) and the reactionwas stirred at r.t. overnight, treated as described in the generalprocedure previously reported to give MST-126 as a white solid in 96.1%yield. m.p 281-283° C.; silica gel TLC R_(f) 0.49 (ethylacetate/petroleum ether 33%); v_(max) (KBr) cm⁻¹, 3334 (N—H urea), 1657(C═O urea), 1592 (aromatic); δ_(H) (400 MHz, DMSO-d₆) 7.27 (2H, s,SO₂NH₂), 7.66 (2H, d, J 8.8, 2×3-H), 7.72 (4H, m, 2×2′-H, 2×3′-H) 7.78(2H, d, J 8.8, 2×2-H), 9.24 (1H, s, NH), 9.26 (1H, s, NH); δ_(C) (100MHz, DMSO-d₆) 153.0 (C═O, urea), 144.0 (m, C-1′), 143.4 (C-4), 138.2(C-1), 127.7 (2×C-2), 127.0 (q, ³J_(C-F) 3.8, C-3′), 125.4 (q, J_(C-F)269, CF₃), 123.0 (q, ²J_(C-F) 32, C-4′), 119.0 (2×C-2′), 118.7 (2×C-3);δ_(F) (376.5 MHz, DMSO-d₆) −60.1 (3F, s).

4-{[([3,5-bis(trifluoromethyl)phenyl]aminocarbonyl)amino]}benzenesulfonamide(MST-127)

4-Aminobenzenesulfanilamide (0.50 g; 2.90 mmols) was treated with3,5-bis(trifluoromethyl)phenylisocyanate (0.74 g; 2.90 mmols) and thereaction was stirred at r.t. overnight, treated as described in thegeneral procedure previously reported to give MST-127 as a white solidin 81.4% yield. m.p 228-229° C.; silica gel TLC R_(f) 0.62 (ethylacetate/petroleum ether 33%); v_(max) (KBr) cm⁻¹, 3374 (N—H urea), 1653(C═O urea), 1596 (aromatic); δ_(H) (400 MHz, DMSO-d₆) H), 7.27 (2H, s,SO₂NH₂), 7.69 (2H, d, J 9.0, 3-H), 7.71 (1H, s, 4′-H), 7.79 (2H, d, J9.0, 2-H), 8.16 (2H, s, 2×2′-H), 9.43 (1H, s, NH), 9.54 (1H, s, NH);δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═O, urea), 143.1, 142.5, 138.5, 131.7(q, ²J_(C-F) 32, 2×C-3′), 127.7 (2×C-2), 124.2 (q, J_(C-F) 272, 2×CF₃),119.2 (m, 2×C-2′), 119.1 (2×C-3), 115.7 (m, C-4′); δ_(F) (376.5 MHz,DMSO-d₆) −61.7 (6F, s).

Ureidosubstituted Compounds (MST-128-133) Materials and Methods

Anhydrous solvents and all reagents were purchased from Sigma-Aldrich,Alfa Aesar and TCI. All reactions involving air- or moisture-sensitivecompounds were performed under a nitrogen atmosphere using driedglassware and syringes techniques to transfer solutions. Infrared (IR)spectra were recorded as KBr plates and are expressed in nhyd⁻¹).Nuclear magnetic resonance (¹H-NMR, ¹³C-NMR, DEPT, HSQC, HMBC) spectrawere recorded using a Bruker Advance III 400 MHz spectrometer in MeOH-d4or in DMSO-d₆. The chemical shifts are reported in parts per million(ppm) and the coupling constants (J) are expressed in Hertz (Hz).Splitting patterns are designated as follows: s, singlet; d, doublet;sept, septet; t, triplet; q, quadruplet; m, multiplet; brs, broadsinglet; dd, double of doubles, appt, aparent triplet, appq, aparentquartet. The assignment of exchangeable protons (OH and NH) wasconfirmed by the addition of D₂O. Analytical thin-layer chromatography(TLC) was carried out on Merck silica gel F-254 plates. Flashchromatography purifications were performed on Merck Silica gel 60(230-400 mesh ASTM) as the stationary phase and ethylacetate/n-hexane orMeOH/DCM were used as eluents. Melting points (mp) were carried out inopen capillary tubes and are uncorrected.

3-(3-(4′-Iodophenyl)ureido)benzenesulfonamide (MST-128)

3-(3-(4′-Iodophenyl)ureido)benzenesulfonamide (A): m.p. 256-258° C.;v_(max) (KBr) cm⁻¹, 3165, 3265, 1643, 1589; δ_(H) (400 MHz, DMSO-d₆)7.34-7.66 (9H, m, Ar—H, SO₂NH₂, exchange with D₂O), 8.10 (1H, d, J, 2.1,2-H), 8.79 (1H, s, NH, exchange with D₂O), 9.08 (1H, s, NH, exchangewith D₂O); δ_(C) (100 MHz, DMSO-d₆) 153.1 (C═O), 145.6, 140.9, 140.3,138.3, 130.3, 122.1, 121.6, 119.9, 116.1, 85.9; Elem. Anal. Calc. [C,37.42; H, 2.90; N, 10.07]. Found [C, 37.06; H, 2.79; N, 9.82]. m/z(ESI⁺) 418 (M+Na)⁺.

3-(3-(4′-Fluorophenyl)ureido)benzenesulfonamide (MST-129)

3-(3-(4′-Fluorophenyl)ureido)benzenesulfonamide (B): m.p. 233-235° C.;v_(max) (KBr) cm⁻¹, 3377, 3352, 1685, 1557; δ_(H) (400 MHz, DMSO-d₆)7.17 (1H, dd, J 8.8, Ar—H), 7.45 (2H, s, SO₂NH₂, exchange with D₂O),7.47-7.60 (5H, m, Ar—H), 8.10 (1H, d, J, 2.1, 2-H), 8.78 (1H, s, NH,exchange with D₂O), 9.04 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz,DMSO-d₆) 158.4 (d, J_(C-F) 237, C-4′), 153.4 (C═O), 145.6, 141.1, 136.6,130.3, 122.0, 121.2, 119.8, 116.3, 116.1; O_(F) (376 MHz, DMSO-d₆)−121.14; Elem. Anal. Calc. [C, 50.48; H, 3.91; N, 13.58]. Found [C,49.98; H, 3.79; N, 13.49]. m/z (ESI⁺) 311 (M+Na)⁺.

3-(3-(3′-Nitrophenyl)ureido)benzenesulfonamide (MST-130)

3-(3-(3′-Nitrophenyl)ureido)benzenesulfonamide (C): m.p. 252-255° C.;v_(max) (KBr) cm⁻¹, 3380, 3350, 1689, 1550; δ_(H) (400 MHz, DMSO-d₆)7.41 (2H, s, SO₂NH₂, exchange with D₂O), 7.48-7.64 (4H, m, Ar—H), 7.77(1H, dd, J 7.2, 2.1, Ar—H), 7.87 (1H, dd, J 7.2, 2.1, Ar—H), 8.14 (1H,d, J, 2.1, 2-H), 8.63 (1H, d, J, 2.1, 2′-H), 9.23 (1H, s, NH, exchangewith D₂O), 9.31 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆)153.3 (C═O), 149.1, 145.7, 141.7, 140.6, 131.0, 130.3, 125.4, 122.4,120.2, 117.4, 116.4, 113.2; Elem. Anal. Calc. [C, 46.43; H, 3.60; N,16.66]. Found [C, 46.91; H, 3.55; N, 16.94]. m/z (ESI⁺) 337 (M+Na)⁺.

3-(3-(4′-Acetylphenyl)ureido)benzenesulfonamide (MST-131)

3-(3-(4′-Acetylphenyl)ureido)benzenesulfonamide (D): m.p. 267-269° C.;v_(max) (KBr) cm⁻¹, 3402, 3351, 2014, 1933, 1912, 1593; δ_(H) (400 MHz,DMSO-d₆) 2.56 (3H, s, CH₃), 7.41 (2H, s, SO₂NH₂, exchange with D₂O),7.48-7.55 (3H, m, 4-H, 5-H, 6-H), 7.60 (2H, d, J 7.2, 2×2′-H), 7.97 (2H,d, J 7.2, 2×3′-H), 8.13 (1H, t, J 2.0, 2-H), 9.18 (1H, s, NH, exchangewith D₂O), 9.19 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆)197.2 (CH₃C═O), 153.0 (C═O), 145.7, 145.0, 140.7, 131.6, 130.5, 130.4,122.2, 120.2, 118.2, 116.2, 27.2; Elem. Anal. Calc. [C, 54.04; H, 4.54;N, 12.60]. Found [C, 54.31; H, 4.47; N, 12.96]. m/z (ESI⁺) 334 (M+Na)⁺.

3-(3-(2′-Isopropylphenyl)ureido)benzenesulfonamide (MST-132)

3-(3-(2′-Isopropylphenyl)ureido)benzenesulfonamide (E): m.p. 175-176°C.; v_(max) (KBr) cm⁻¹, 3328, 3300, 1690, 1556; δ_(H) (400 MHz, DMSO-d₆)1.23 (6H, d, J 6.2, 2×8′-H₃), 3.19 (1H, sept, J 6.2, 7′-H), 7.14 (2H, m,Ar—H), 7.35 (1H, d, J 7.2, Ar—H), 7.37 (2H, s, SO₂NH₂, exchange withD₂O), 7.59-7.70 (4H, m, Ar—H), 8.00 (1H, s, NH, exchange with D₂O), 8.10(1H, t, J 2.0, 2-H), 9.30 (1H, s, NH, exchange with D₂O); δ_(C) (100MHz, DMSO-d₆) 153.9 (C═O), 145.6, 141.3, 140.7, 136.1, 130.3, 126.7,126.2, 125.2, 124.7, 121.7, 119.5, 115.8, 27.8, 24.0; Elem. Anal. Calc.[C, 57.64; H, 5.74; N, 12.60]. Found [C, 58.14; H, 5.73; N, 12.70]. m/z(ESI⁺) 334 (M+Na)⁺.

3-(3-(Perfluorophenyl)ureido)benzenesulfonamide (MST-133)

3-(3-(Perfluorophenyl)ureido)benzenesulfonamide (F): m.p. 224-227° C.;V_(max) (KBr) cm⁻¹, 3390, 3287, 1785, 1560; δ_(H) (400 MHz, DMSO-d₆)7.38 (2H, s, SO₂NH₂, exchange with D₂O), 7.50 (2H, m, Ar—H), 7.63 (1H,d, J 7.2, Ar—H), 8.09 (1H, s, 2-H), 8.63 (1H, s, ArNHCONH, exchange withD₂O), 9.47 ((1H, s, ArNHCONH, exchange with D₂O); δ_(C) (100 MHz,DMSO-d₆) 152.9 (C═O), 145.6, 144.1 (d, J¹ _(C-F) 245), 140.7, 139.6 (d,J¹ _(C-F) 253), 138.2 (d, J¹ _(C-F) 245), 130.4, 122.3, 120.4, 116.4,114.7 (t, J_(C-F) 12); δ_(F) (376 MHz, DMSO-d₆) −146.3 (2F, dd, J 19.24.8, 2×2′-F), −159.2 (1 F, t, J 22.9, 4′-F), −164.0 (2F, dt, J 22.6 4.8,2×3′-F); Elem. Anal. Calc. [C, 40.95; H, 2.11; N, 11.02]. Found [C,40.68; H, 1.74; N, 11.00]. m/z (ESI⁺) 382 (M+Na)⁺.

TABLE 2 CA INHIBITION DATA WITH META-UREIDOSUBSTITUTED SULFONAMIDESMST-128 TO -133. Ki (nM) Compound hCA I hCA II hCA IX hCA XII MST-128426 67 13.1 4.5 MST-129 414 59 10.2 5.8 MST-130 614 41 7.9 8.2 MST-131762 74 15.1 12.8 MST-132 593 38 18.5 13.7 MST-133 69 5.4 4.2 4.8

Synthesis of Ureidosulfonamides Corresponding to MST-134-1484-(3-(4′-chloro-2-fluorophenyl)ureido)benzenesulfonamide MST-134

4-(3-(4′-Chloro-2-fluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.26-7.30 (3H,brs, SO₂NH₂, exchange with D₂O, 5′/6′-H), 7.52 (1H, dd, J, 8.2, 2.1,5′/6′-H), 7.64 (2H, d, J 8.2, 2×⅔-H), 7.78 (2H, d, J 8.2, 2×⅔-H), 8.19(1H, dd, J 9.0, 8.2 3′-H), 8.79 (1H, s, NH, exchange with D₂O), 9.47(1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 152.8 (d, J¹_(C-F) 245, C-2′), 152.7 (C═O), 143.2, 138.2, 127.8, 127.4, (d, J_(C-F)10), 126.7 (d, J_(C-F) 10), 125.6, 122.5, 118.4, 116.6 (d, J_(C-F) 23);δ_(F) (376 MHz, DMSO-d₆) −126.38.

4-(3-(4′-bromo-2′-fluorophenyl)ureido)benzenesulfonamide MST-135

4-(3-(4′-Bromo-2′-fluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.27 (2H, s,SO₂NH₂, exchange with D₂O), 7.40 (1H, d, J, 9.2 5′/6′-H), 7.64 (3H, m,2×⅔-H, 5′/6′-H), 7.78 (2H, d, J 9.2, 2×⅔-H), 8.16 (1H, t, J 8.8 3′-H),8.79 (1H, s, NH, exchange with D₂O), 9.48 (1H, s, NH, exchange withD₂O); δ_(C) (100 MHz, DMSO-d₆) 152.8 (d, J¹ _(C-F) 245, C-2′), 152.7(C═O), 143.2, 138.2, 128.5, 127.9, 127.7 (d, J_(C-F) 6), 122.8, 119.2(d, J_(C-F) 22), 118.4, 114.0 (d, J_(C-F) 9); δ_(F) (376 MHz, DMSO-d₆)−126.38.

4-(3-(2′-fluoro-5-nitrophenyl)ureido)benzenesulfonamide MST-136

4-(3-(2′-Fluoro-5′-nitrophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.28 (2H, s,SO₂NH₂, exchange with D₂O), 7.60 (1H, dd, J, 9.2 6.8, 6′-H), 7.70 (2H,d, J 9.2, 2×⅔-H), 7.80 (2H, d, J 9.2, 2×⅔-H), 7.96 (1H, m, 4′-H), 9.13(1H, s, NH, exchange with D₂O), 9.18 (1H, m, 6′-H), 9.57 (1H, s, NH,exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 156.0 (d, J¹ _(C-F) 251,C-2′), 154.8 (C═O), 144.9, 142.9, 138.6, 129.3 (d, J_(C-F) 12), 127.9,119.0 (d, J_(C-F) 9), 118.7, 117.0 (d, J_(C-F) 22), 115.7; δ_(F) (376MHz, DMSO-d₆) −119.43

4-(3-(2′,4′,5′-trifluorophenyl)ureido)benzenesulfonamide MST-137

4-(3-(2′,4′,5′-Trifluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.27 (2H, s,SO₂NH₂, exchange with D₂O), 7.68 (3H, m, 2×⅔-H, 3′-H), 7.78 (2H, d, J9.2, 2×⅔-H), 8.22 (1H, m, 6′-H), 8.58 (1H, s, NH, exchange with D₂O),9.47 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 152.7(C═O), 148.2 (dd, J¹ _(C-F) 240, 12.2), 146.4 (d, J¹ _(C-F) 237, 15.8),144.4 (dd, J¹ _(C-F) 250, 12.0), 143.3, 138.3, 127.8, 125.0 (m), 118.5,109.5 (dd, J_(C-F) 24.5, 2.9), 106.4 (dd, J_(C-F) 25.6, 22.0); δ_(F)(376 MHz, DMSO-d₆) −130.64 (d, J_(F-F) 13.9), −141.75 (m), −143.06 (d,J_(F-F) 24.4).

4-(3-(2′-fluoro-5′-(trifluoromethyl)phenyl)ureido)benzenesulfonamideMST-138

4-(3-(2′-Fluoro-5′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide:V_(max) (KBr) cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆)7.28 (2H, s, SO₂NH₂, exchange with D₂O), 7.47 (1H, m, 4′-H), 7.55 (1H,dd, J 10.8 8.8, 3′-H), 7.67 (2H, d, J 8.8, 2×⅔-H), 7.79 (2H, d, J 8.8,2×⅔-H), 8.63 (1H, m, 6′-H), 9.03 (1H, s, NH, exchange with D₂O), 9.56(1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 154.0 (d, J¹_(C-F) 247, C-2′), 152.8 (C═O), 143.0, 138.5, 129.30 (d, J_(C-F) 11.3),127.9, 126.2 (dd, J_(C-F) 31.8, 3.3), 123.5, 120.7 (m), 118.6, 117.7,117.0 (d, J_(C-F) 20.5); δ_(F) (376 MHz, DMSO-d₆) −60.7 (3F, C—F₃),−123.8 (1F, 2′-F).

4-(3-(2′-fluoro-3′-(trifluoromethyl)phenyl)ureido)benzenesulfonamideMST-139

4-(3-(2′-fluoro-3′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide:V_(max) (KBr) cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆)7.27 (2H, s, SO₂NH₂, exchange with D₂O), 7.43 (2H, m, 5′-H, 6′-H), 7.65(2H, d, J 8.8, 2×⅔-H), 7.79 (2H, d, J 8.8, 2×⅔-H), 8.46 (1H, m, 4′-H),8.96 (1H, s, NH, exchange with D₂O), 9.53 (1H, s, NH, exchange withD₂O); δ_(C) (100 MHz, DMSO-d₆) 152.7 (C═O), 150.0 ((d, J¹ _(C-F) 250,C-2′), 143.1, 138.3, 129.4 ((d, J_(C-F) 9), 127.8, 125.9 (d, J_(C-F)43.9), 124.9, 122.2, 120.4, 118.7, 117.5 (dd, J_(C-F) 32.1, 3.2); δ_(F)(376 MHz, DMSO-d₆) −59.8 (3F, d, J_(F-F) 13.2, C—F₃), −132.2 (1F, q, J13.2, 2′-F).

4-(3-(2′,3′,4′-trifluorophenyl)ureido)benzenesulfonamide MST-140

4-(3-(2′,3′,4′-Trifluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.27 (2H, s,SO₂NH₂, exchange with D₂O), 7.34 (2H, m, 5′/6′-H), 7.65 (2H, d, J 8.8,2×⅔-H), 7.77 (2H, d, J 8.8, 2×⅔-H), 7.88 (1H, m, 5′/6′-H), 8.82 (1H, s,NH, exchange with D₂O), 9.45 (1H, s, NH, exchange with D₂O); δ_(C) (100MHz, DMSO-d₆) 152.9 (C═O), 146.6 (d, J¹ _(C-F) 237), 143.2, 141.8 (d, J¹_(C-F) 240), 139.6 (d, J¹ _(C-F) 242), 138.3, 127.8, 126.0 (m), 118.5,116.7 (m), 112.6 (dd, J_(C-F) 18, 4); δ_(F) (376 MHz, DMSO-d₆) −143.1(1F, d, J_(F-F) 21.7, 2′/4′-F), −148.4 (1F, d, J_(F-F) 21.7, 2′/4′-F),−161.1 (1F, t, J_(F-F) 21.7, 3′-F).

4-(3-(2′-fluorophenyl)ureido)benzenesulfonamide MST-141

4-(3-(2′-Fluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr) cm⁻¹,3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.07 (1H, m, 3′-H),7.20 (1H, m, 4′-H), 7.26 (3H, brs, SO₂NH₂, exchange with D₂O, 5′-H),7.64 (2H, d, J 8.8, 2×⅔-H), 7.80 (2H, d, J 8.8, 2×⅔-H), 8.20 (1H, m,6′-H) 8.70 (1H, s, NH, exchange with D₂O), 9.46 (1H, s, NH, exchangewith D₂O); δ_(C) (100 MHz, DMSO-d₆) 153.1 (d, J¹ _(C-F) 240), 152.9(C═O), 143.4, 138.1, 128.1, 127.8, 125.4, 123.8, 121.7, 118.3, 116.6 (d,J_(C-F) 19); δ_(F) (376 MHz, DMSO-d₆) −129.59.

4-(3-(2′,4′-difluorophenyl)ureido)benzenesulfonamide MST-142

4-(3-(2′,4′-Difluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.08 (1H, m,3′/5′-H), 7.25 (2H, s, SO₂NH₂, exchange with D₂O), 7.36 (1H, m,3′/5′-H), 7.64 (2H, d, J 8.8, 2×⅔-H), 7.78 (2H, d, J 8.8, 2×⅔-H), 8.11(1H, m, 6′-H), 8.64 (1H, s, NH, exchange with D₂O), 9.41 (1H, s, NH,exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 158.1 (dd, J_(C-F) 243,13), 153.4 (dd, J_(C-F) 244, 12), 153.0 (C═O), 143.4, 138.1, 128.1,127.8, 124.5 (d, J_(C-F) 9), 123.2 (d, J_(C-F) 8), 118.4, 112.0 (d,J_(C-F) 21), 104.8 (t, J_(C-F) 25); δ_(F) (376 MHz, DMSO-d₆) −117.52,−124.3.

4-(3-(3′-chlorophenyl)ureido)benzenesulfonamide MST-143

4-(3-(3′-chlorophenyl)ureido)benzenesulfonamide: v_(max) (KBr) cm⁻¹,3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.07 (1H, d, J 8.8,4′-H), 7.25 (2H, s, SO₂NH₂, exchange with D₂O), 7.33 (2H, m, 5′-H,6′-H), 7.64 (2H, d, J 8.8, 2×⅔-H), 7.77 (3H, m, J 8.8, 2×⅔-H, 2′-H),9.04 (1H, s, NH, exchange with D₂O), 9.17 (1H, s, NH, exchange withD₂O); δ_(C) (100 MHz, DMSO-d₆) 153.1 (C═O), 143.5, 141.8, 138.0, 134.1,131.3, 127.7, 122.7, 118.7, 118.6, 117.8.

4-(3-(2′,5′-dichlorophenyl)ureido)benzenesulfonamide MST-144

4-(3-(2′,5′-Dichlorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3166, 3270, 1640, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.15 (1H, dd, J8.8 2.8, 4′-H), 7.28 (2H, s, SO₂NH₂, exchange with D₂O), 7.55 (1H, dd, J8.8 2.8, 3′-H), 7.64 (2H, d, J 8.8, 2×⅔-H), 7.79 (2H, d, J 8.8, 2×⅔-H),8.35 (1H, d, J 2.8, 6′-H), 8.60 (1H, s, NH, exchange with D₂O), 9.90(1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 152.7 (C═O),143.1, 138.4, 137.8, 132.9, 131.5, 127.9, 124.0, 121.3, 121.1, 118.7.

4-(3-(2′-Chloro-5-nitrophenyl)ureido)benzenesulfonamide MST-145

4-(3-(2′-Chloro-5′-nitrophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3164, 3271, 1641, 1592; δ_(H) (400 MHz, DMSO-d₆) 7.29 (2H, s,SO₂NH₂, exchange with D₂O), 7.70 (2H, d, J 8.8, 2×⅔-H), 7.82 (3H, m,2×⅔-H, 3′-H), 7.93 (1H, dd, J 8.9 2.2, 4′-H), 8.84 (1H, s, NH, exchangewith D₂O), 9.20 (1H, d, J 2.2, 6′-H), 9.99 (1H, s, NH, exchange withD₂O); δ_(C) (100 MHz, DMSO-d₆) 152.7 (C═O), 147.5. 142.9, 138.6, 137.7,131.3, 128.9, 127.9, 118.8, 118.5, 115.6.

4-(3-(2′-Chloro-4′-(trifluoromethyl)phenyl)ureido)benzenesulfonamideMST-146

4-(3-(2′-Chloro-4′-(trifluoromethyl)phenyl)ureido)benzenesulfonamide:v_(max) (KBr) cm⁻¹, 3169-1639, 1560; δ_(H) (400 MHz, DMSO-d₆) 7.29 (2H,s, SO₂NH₂, exchange with D₂O), 7.69 (2H, d, J 8.8, 2×⅔-H), 7.74 (1H, dd,J 8.8 4.0, 5′-H), 7.80 (2H, d, J 8.8, 2×⅔-H), 7.92 (1H, dd, J 4.0,3′-H), 8.50 (1H, d, J 8.8, 6′-H), 8.76 (1H, s, NH, exchange with D₂O),10.0 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 152.5,143.0, 140.4, 138.5, 128.7, 127.9, 127.2 (m), 125.8 (m), 124.0 (d,J_(C-F) 33.0), 13.2, 122.7, 121.4; δ_(F) (376 MHz, DMSO-d₆) −60.42.

4-(3-(2′,6′-difluorophenyl)ureido)benzenesulfonamide MST-147

4-(3-(2′,6′-Difluorophenyl)ureido)benzenesulfonamide: v_(max) (KBr)cm⁻¹, 3164, 3270, 1641, 1590; δ_(H) (400 MHz, DMSO-d₆) 7.33 (4H, m,SO₂NH₂, exchange with D₂O, 2×5′-H), 7.37 (1H, m, 4′-H), 7.64 (2H, d, J8.8, 2×⅔-H), 7.76 (2H, d, J 8.8, 2×⅔-H), 8.30 (1H, s, NH, exchange withD₂O), 9.38 (1H, s, NH, exchange with D₂O); δ_(C) (100 MHz, DMSO-d₆) 158(d, J¹ _(C-F) 247, C-2′, C-6′), 153.27 (C═O), 143.7, 138.0, 128.3 (m),127.7, 118.5, 115.9 (t, J_(C-F) 16, C-4′), 112.6 (d, J_(C-F) 23,2×C-3′); δ_(F) (376 MHz, DMSO-d₆) −118.73.

4-(3-(perchlorophenyl)ureido)benzenesulfonamide MST-148

4-(3-(perchlorophenyl)ureido)benzenesulfonamide: v_(max) (KBr) cm⁻¹,3168, 3275, 1641, 1590; δ_(H) (400 MHz, DMSO-d₆) 7.24 (2H, m, SO₂NH₂,exchange with D₂O), 7.64 (2H, d, J 8.8, 2×⅔-H), 7.75 (2H, d, J 8.8,2×⅔-H), 8.29 (1H, s, NH, exchange with D₂O), 9.38 (1H, s, NH, exchangewith D₂O); δ_(C) (100 MHz, DMSO-d₆) 153.2 (C═O), 142.0, 139.2, 130.1(overlapping signals), 127.4, 125.5.

TABLE 3 CA INHIBITION DATA WITH UREIDOSUBSTITUTED SULFONAMIDESMST-134-148 Ki (nM) Compound hCA I hCA II hCA IX hCA XII MST-134 436 1625.1 7.9 MST-135 373 418 6.3 7.4 MST-136 519 276 7.3 7.6 MST-137 569 2157.6 6.2 MST-138 363 170 7.4 10.1 MST-139 395 196 8.7 8.5 MST-140 484 3359.6 6.4 MST-141 550 33.0 7.7 7.3 MST-142 275 60.6 3.7 7.1 MST-143 15810.0 8.4 8.5 MST-144 747 58.2 8.9 6.8 MST-145 307 0.94 8.2 6.1 MST-146192 0.97 8.4 8.2 MST-149 158 0.93 6.1 8.3 MST-150 538 362 6.3 7.6

For the remaining examples, the following methods and additionalinformation will be a useful reference.

Cell Culture and Hypoxic Exposure

The acquisition, generation and culture of the luciferase expressingmouse breast cancer cell lines 4T1, 66cl4 and 67NR, and the human breastcancer cell lines MDA-231 and MDA-231 LM2-4 have been describedpreviously (Lou et al, (2008) Dev Dyn 237:2755-2768; Lou et al, (2011)Cancer Res, 71:3364-3376. For culture in hypoxia, cells were maintainedin 1% 02 and 5% CO2 balanced with N2 at 37° C. in a humidified incubatorin a sealed anaerobic workstation.

Generation of Stable Cells

shRNAmir vectors targeting mouse CAIX and a non-silencing sequence (OpenBiosystems) were transfected into 90% confluent cells usingLipofectAMINEPLUS™ (Invitrogen Life Technologies) according to themanufacturer's instructions. Due to the previous utilization ofpuromycin, transfected cells were selected using hygromycin. StableshCAIX clones were derived by limited dilution cloning. For(re-)introduction of CAIX into cells, human CAIX (gift from Dr. JacquesPouyssegur, University of Nice) was transfected into 4T1 cells followingthe same procedure and Zeocin was used for selection.

Measurement of Extracellular pH

Changes in solution pH were assessed using procedures publishedpreviously (29, 42, 43). In brief, cells were plated at appropriatedensity (1×104 cells/cm2 for 4T1 cells and its transfected derivatives,2×104 cells/cm2 for 66cl4 cells, 1×104 cells/cm2 for 67NR cells and itstransfected derivatives) in 60 mm dishes and allowed to recoverovernight. A standard volume of 3 ml of fresh media/dish was then addedand cells were incubated in normoxia (air+5% CO2) or hypoxia (1% O2 and5% CO2 balanced with nitrogen) for 72 h. Care was taken to ensure thatcultures grown in normoxia and hypoxia were at similar confluence andcontained similar cell numbers at the time of medium collection.Collected spent media was maintained at 37° C. and pH was measuredimmediately using a digital pH meter. Cell counts were performed toensure that cell numbers for a given cell line were comparable in bothenvironmental conditions. Cells were harvested on ice for qRT-PCR andWestern blot analysis.

Pharmacological Inhibitors

The chemical properties of the sulfonamide, MST-017, have been describedpreviously under the name CAI 17 (Supuran, C. 2008, Nature. Vol 7:168-181). Ureido sulfonamides are new. For in vitro studies, thecompounds were dissolved in DMSO, stored at −80° C. and diluted intoculture medium just prior to application. Subconfluent cells wereincubated with MST-017 for 72 hours in normoxia or hypoxia, washed 3× inPBS and imaged using a Zeiss Axioplan™ epifluorescence microscope. Forin vivo studies, the MST-017 inhibitor was administered by i.p.injection (first two doses were administered i.v.) at 75 mg/kg and 150mg/kg 3× per week for 2 weeks. Dosing concentrations and schedules forthe other inhibitors are indicated in the appropriate examples below.The compounds were solubilized in PEG400/ethanol/saline prior toinjection. Vehicle components were held constant as inhibitorconcentrations were varied.

Analysis of mRNA and Protein Expression

Quantitative Real-Time PCR (qRT-PCR) was conducted in 384-well plates onan Applied Biosystems instrument using a Roche Universal Probe Library(UPL) according to the manufacturer's instructions. Briefly, 1 μg oftotal RNA from either subconfluent cells or snap frozen tissue was usedto make cDNA. 10 μl of qRT-PCR mixture containing 100 nM UPL probe, 200nM of each primer (Invitrogen) and TaqMan™ PCR master mix (AppliedBiosystems) was loaded into each well for 40 cycles of PCR (44).Relative gene expression quantification data were acquired and analyzedusing an ABI Prism 7900HT Sequence Detection System and the standard2-ΔΔct method using β-actin as the housekeeping gene. Forimmunoblotting, cells or flash frozen tumor tissue were lysed in 1%Triton X-100 buffer (50 mM Hepes, pH=7.5, 150 mM NaCl, 10% glycerol, 1mM EGTA and 2 mM EDTA), supplemented with the appropriate inhibitors.Equal amounts of protein were loaded on SDA-PAGE gels. To enhance thedetection of HIF-1α before degradation, cells at equal densities weredirectly lysed in 4×SDS loading buffer in hypoxia. Western blots wereperformed using mouse CAIX (1:500), HIF-1α (1:250), human CAIX (1:1000)(all from R&D Systems) and β-actin (1:10,000, Sigma) antibodies.

Mouse Tumor Models

All animal studies and procedures were done in accordance with protocolsapproved by the Institution Animal Care Committee at the BC CancerResearch Centre and the University of British Columbia (Vancouver, BC,Canada).

Syngeneic Orthotopic Tumors and Spontaneous Metastasis

4T1 cells (1×10⁶) or 67NR cells (2×10⁶) were orthotopically implantedinto the fourth mammary fat pad of 7-9 week-old female BALB/c mice asdescribed previously (Lou et al, (2011) Cancer Res 71:3364-3376; Lou etal, (2008) Dev Dyn 237:2755-2768). Injection of cell numbers of thismagnitude is standard for propagation of these tumors, and is well belowthat used in other models of tumor growth (Erler, J T. Bennewith, K L,Icolau, M. Nature 440: 1222-1226). Primary tumor growth rates werecalculated from caliper measurements using the modified ellipsoidformula (L×W²)/2. Tumor formation and metastasis progression wasmonitored and quantified using bioluminescent imaging as previouslydescribed (Ebos et al., (2009) Cancer Cell 15:232-239; Lou et al.,(2008) Dev Dyn 237:2755-2768).

Experimental Metastasis Assays

For studies involving genetic depletion of CAIX, 4T1 or 67NR cells(5×10⁵) were injected directly into the tail vein of 7-9 week-old femaleBALB/c mice. Mice were imaged once per week to follow the growth ofmetastases. Mice were euthanized 20 days post-injection and lungs wereresected for further analysis. Tumor burden in the lung was quantifiedby manually counting nodules visible on the lung surface. For studiesusing sulphonamide inhibitors, 4T1 cells (1-5×10⁵) were injected asdescribed above (Pacchiano et al, (2011) J Med Chem 54:1896-1902).

Human Xenograft Tumors

For studies involving CAIX depletion, 1×10⁷ MDA-MB-231 cells suspendedin a 50% Matrigel/PBS solution were implanted subcutaneously in 6-8week-old female NOD.CB17-prkdc^(scid)/J mice. For primary breast tumorxenografts using the MDA-MB-231 LM2-4^(Luc+) variant (Ebos et al, (2009)Cancer Cell 15:232-239), 2×10⁶ cells were implanted orthotopically inmice as described above. Therapy was initiated when the tumors reached200 mm³. For both models, tumor growth was monitored by calipermeasurement.

3D Matrigel Invasion Assay

A 3D “on-top” matrigel culture assay was performed as describedpreviously (Lee et al, (2007) Nat Methods 4:359-365). Briefly, MDA-231LM2-4 Luc+ cells (1.5×10⁴ cells/cm²) were resuspended in 100 μl/wellgrowth media containing 2× the final concentration of inhibitor andplated into 8-well chamber slides precoated with matrigel. Cells wereallowed to attach for 45 minutes with side-to-side agitation every 10-15minutes to prevent clumping of cells in the center of the well. Anadditional 100 μl/well media containing 10% matrigel was added to thecells and cultures were incubated in hypoxia for 4 days. Images wereacquired and cultures were fixed for TUNEL using the “whole culturefixation” methodology outlined in Lee et al, (2007) Nat Methods4:359-365).

Tumorsphere Culture

4T1 cells were grown as monolayers with twice weekly sub-cultivation inDMEM (Gibco) containing 5% fetal bovine serum (FBS) (Sigma).Subsequently, shNS and shCAIX 4T1 were cultured as tumorspheres inmammocult media (StemCell Technologies, Vancouver, B. C., Canada) as perthe manufacturer's instructions.

(Tumorspheres are 3-dimensional structures (often spherical in shape)composed of adherent cancer cells that form when tumor cells arecultured in vitro under specific growth conditions (Fillmore andKuperwasser, (2008) Breast Cancer Res. 10: R25). Tumorspheres generallygrow in suspension culture and are considered the in vitro surrogate toin vivo tumors.)

Flow Cytometric Analyses

4T1 tumorspheres were incubated with trypsin, washed once in HF buffer(HBSS containing 2% Fetal Bovine Serum), then stained with anti-CD24-APCand anti-CD44-PECy7 using 0.3 μl of antibody per 106 cells in 100 ul HF,and incubated on ice for 10 min. Following incubation, cells were washedonce with HF buffer and resuspended in 300 ul HF buffer containing4′,6-diamidino-2-phenylindole (DAPI; final concentration, 1 μg/ml).Cells were separated on an Aria cell sorter (BD Biosciences, San Jose,Calif., USA). Live cells were gated on the basis of forward and sidescatter, and single cells were gated on the basis of forward scatter andpulse width. Gates were determined by analysis of unstained cells,isotype specific controls, and single stains. The CD44+CD24−/low orCD44+CD24+ cells were not assessed for purity due to the low numbers ofcells obtained. The cell counter of the flow cytometers was used todetermine cell numbers. Cells were collected into DMEM media or HFbuffer.

Immunohistochemistry

Two hours before tumor excision mice were injected i.p. with a salinesolution containing 1500 mg/kg BrdUrd (Sigma) and 60 mg/kg Pimonidazole(Chemicon), and i.v. 5 min before with DiOC7(3) (70 μl, 0.6 mg/ml;Molecular Probes). Serial tumor cryosections (10 μm) were cut with aCryostar™ HM560 (Microm International), air dried for 24 h, and imagedfor DiOC7(3) tissue fluorescence to visualize blood flow. Sections werefixed in 50% (v/v) acetone/methanol for 10 min at room temperature. Thestaining was performed using anti-PECAM/CD31 antibody (1:2000 clone,2H8, BD Pharmingen) and Alexa 647 anti-hamster secondary (1:200,Invitrogen) for vasculature, polyclonal rabbit-anti-pimonidazole(1:2000, Hydroxyprobe Inc.) and Alexa 488 anti-rabbit secondary (1:200,Molecular Probes) for hypoxia, TUNEL (Roche Diagnostics) with a TMR redtagged dUTP for apoptosis. After fluorescence imaging slides weretransferred to distilled water for 10 min followed by 1 h treatment with2 M HCl and 5 min neutralization with 0.1 M sodium borate. DNAincorporated BrdUrd was detected using monoclonal rat anti-BrdUrd(1:500, clone BU1/75, Sigma) and anti-mouse peroxidase conjugateantibody (1:200, Sigma) and a metal enhanced DAB substrate (1:10,Pierce). haematoxylin counterstained slides were dehydrated and mountedusing Permount (Fisher Scientific) before imaging. Image acquisition andanalysis was done as previously described (Kyle, A H., Huxham, L A.,Yeoman, D M., et al 2007. Clin Cancer Res 13:2804-2810). Paraffinembedded tumor sections were also stained for CAIX (1:100 for primarytumors, 1:50 for lung metastases, Santa Cruz Biotechnology) and HIF-1α(1:100, R&D Systems) as previously described (Luo et al.). 26 For thelymphangiogenesis studies, frozen tissue sections were fixed with 2% PFAfor 20 min, and stained with rabbit anti-LYVE-1 (1:100, R&D Systems) andrat anti-CD31 (1:100, BD Pharmigen) dissolved in PBS containing 10%bovine serum albumin and 2% goat serum for 1 h at room temperature in ahumidified container. Alexa 488 anti-rabbit and Alexa 546 anti-ratantibodies were used as secondary antibodies for 1 h followed byVectashield mounting medium (Vector Laboratories) containing DAPInuclear counter stain for mounting.

Cell Proliferation Assay

Cell growth was measured using an MTT cell proliferation kit (RocheApplied Science) according to the manufacturer's instructions. In brief,cells were plated in 96-well plates at a density of 5×103 cells/cm2 andallowed to recover overnight. Parallel samples were then incubated innormoxia and hypoxia for 48 to 72 h prior to performing the assay.

Apoptosis Assay

TUNEL labeling (Roche Applied Science) was employed for analysis ofapoptosis mostly according to the manufacturer's instructions. Briefly,subconfluent cells grown on coverslips were incubated for 48 h undernormoxia or hypoxia in 1% serum, air-dried, fixed in 4% paraformaldehydefor 60 min and permeabilized for 10 min in PBS and 0.1% Triton-X-100 atroom temperature. Cell layers were 27 then incubated with the TUNELreagents for 60 min at 37° C., washed in PBS and counterstained with a1:10,000 dilution of H33342.

Historical Clinical Analysis

A tissue microarray of 4,444 patients with a new diagnosis of invasivebreast cancer in the province of British Columbia from 1986 to 1992 wascreated from tumor specimens submitted to a central estrogen receptorlaboratory. The methods used to create the TMAs have been described(Cheang, M D., Chia, S K., Vodu, D et al. 2009. J Natl Cancer Inst101:736-750). The TMA cohort representing were approximately 70% of allbreast cancer cases diagnosed during this time were all referred to theBritish Columbia Cancer Agency. 3,630 cases had adequate tumor andstaining results for assessment of all biomarkers. Immunohistochemistryfor ER, PR, HER2, CK 5/6, EGFR and Ki67 was performed concurrently onserial sections and scored as described previously (Cheang M D et al.).CAIX expression was assessed using a murine monoclonal antibody (M75;1:50) (Choi, S W., Kim, J Y., Park, J Y. 2008 Hum Pathol 39:1317-1322).Scoring of CAIX expression was either 0: no staining or 1: any stainingand performed independently and blindly by 2 pathologists. Priorapproval of the study was obtained from the Ethics Committee of theUniversity of British Columbia.

Statistical Analysis

Results were subjected to statistical analysis using the Data AnalysisToolPack™ in Excel software. Two-tailed p values were calculated usingstudent's t-test. Data were considered significant for p<0.05.Statistical analysis for the clinical outcomes was performed using SPSS13.0 (Chicago, Ill.), S-Plus 6.2 (Seattle, Wash.) and R 2.1.1(http://www.r-project.org). In univariate analysis, BCSS (date ofdiagnosis of primary breast cancer to date of death with breast canceras the primary or underlying cause) and RFS (date of diagnosis ofprimary breast cancer to the date of a local, regional or distantrecurrence) and distant RFS (date of diagnosis of primary breast cancerto the date of a distant recurrence) were estimated by Kaplan-Meiercurves. Log-rank test was used to estimate the survival differences. Formultivariate analysis, a Cox proportional hazards model was used toestimate the adjusted hazard ratios and significance. To assess theviolations of proportional hazard models, smoothed plots of weightedSchoenfeld residuals were used.

Example 2 CAIX is a Prognostic Marker in a Large Cohort of Breast CancerPatients

Although previous studies have reported that CAIX expression in severaltypes of cancer, including breast cancer, correlates with poor patientprognosis as previously described, the sample sizes have been relativelysmall and adjuvant treatments not uniform. To validate CAIX as animportant prognostic marker in a large sample population subjected tostandardized treatment, we analyzed the expression of CAIX in a primarybreast tumor tissue microarray (TMA) containing 3992 patient sampleswith a median follow-up of 10.5 years.

The methods used to create the TMAs have been described (Cheang, M D.,Chia, S K., Vodu, D et al. 2009. J Natl Cancer Inst 101:736-750). TheTMA cohort representing were approximately 70% of all breast cancercases diagnosed during this time were all referred to the BritishColumbia Cancer Agency. CAIX expression was assessed using a murinemonoclonal antibody (M75; 1:50) (Choi, S W., Kim, J Y, Park, J Y. 2008Hum Pathol 39:1317-1322). Scoring of CAIX expression was either 0: nostaining or 1: any staining and performed independently and blindly bytwo pathologists. Prior approval of the study was obtained from theEthics Committee of the University of British Columbia.

Statistical Analysis

Results were subjected to statistical analysis using the Data AnalysisToolPack™ in Excel software. Two-tailed p values were calculated usingstudent's t-test. Data were considered significant for p<0.05.Statistical analysis for the clinical outcomes was performed using SPSS13.0 (Chicago, Ill.), S-Plus 6.2 (Seattle, Wash.) and R 2.1.1(http://www.r-project.org). In univariate analysis, BCSS (date ofdiagnosis of primary breast cancer to date of death with breast canceras the primary or underlying cause) and RFS (date of diagnosis ofprimary breast cancer to the date of a local, regional or distantrecurrence) and distant RFS (date of diagnosis of primary breast cancerto the date of a distant recurrence) were estimated by Kaplan-Meiercurves. Log-rank test was used to estimate the survival differences. Formultivariate analysis, a Cox proportional hazards model was used toestimate the adjusted hazard ratios and significance. To assess theviolations of proportional hazard models, smoothed plots of weightedSchoenfeld residuals were used.

CAIX expression was seen in 15.6% of assessable tumors and CAIX wasdifferentially expressed among the biological subtypes, with the highestcorrelation in the basal breast cancers (51%) and the lowest proportionin the luminal A subtype (8%) (Table 5 below).

In Kaplan-Meier analyses, CAIX expression was significantly associatedwith worse relapse free survival (FIG. 1A), distant relapse freesurvival (FIG. 1B) and breast cancer specific survival (FIG. 1C),achieving very high levels of statistical significance (p<10-17,p<10-16, and p<10-13, respectively). The 10 year distant relapse freesurvival and breast cancer specific survival rates in the CAIX positiveversus CAIX negative groups were 57% compared to 73%, and 62% comparedto 78%, respectively. In multivariate analyses, including all standardprognostic variables and biological subtypes, CAIX expression remained astrong independent poor prognostic factor with a hazard ratio of 1.4.These data confirm and extend the results of previous studies that haveshown that CAIX is a prognostic marker in a large number of breastcancer patients.

This example provides evidence that the compounds disclosed herein willbe therapeutic for any cancers susceptible to metastases, or thoseoverexpressing CAIX, a target shown in a large patient databank to beassociated with decreased survival in patients.

TABLE 4 CAIX EXPRESSION ACCORDING TO BIOLOGICAL SUBTYPE N CAIX % CAIXBreast Cancer Subtype Total N +ve +ve LumA (ER or PR+, HER2−, ki67−)1437 120 8% LumB (ER or PR+, Her2−, ki67+) 815 88 11% Lum/HER2+ (Her2+,ER or PR+) 213 36 17% Her2+ (Her2+, ER−, PR−) 239 80 33% Basal (ER−,PR−, Her2−, CK56 or 327 168 51% EGFR +) Above, LumA is luminal A; LumBis luminal B; ER is estrogen receptor; PR is progesterone receptor; EGFRis epithelial growth factor receptor; and “+ve” is positive.

Example 3 Metastatic 4T1 Tumors are Characterized by Hypoxia and CAIXExpression

FIG. 2 shows cell cultures, mouse models with bioluminescent labeling,and a hypoxia-induced gene expression table for three tumor cell lines.Briefly, metastatic (4T1, 66cl4) and non-metastatic (67NR) mouse mammarytumor cell lines stably expressing luciferase were inoculated into themammary fat pad of mice. Tumor formation and metastatic progression weremonitored by bioluminescent imaging. Primary tumor cells were isolatedby laser microdissection and differential gene expression analysis wasperformed on isolated tumor cells. (B) Tumor tissue from three mice fromeach cell model was analyzed for expression of hypoxia-induced genes(high expression, dark; low expression, light).

Non-metastatic 67NR tumors exhibited high vascular density, were largelydevoid of hypoxia, and had low numbers of apoptotic cells. In contrast,primary tumors derived from the metastatic cell lines, especially the4T1 cell line, were poorly vascularized, and had large areas of hypoxiaand necrosis with high numbers of apoptotic cells (FIG. 2). Among thehypoxia-inducible genes identified in these tumors, the expression ofCAIX, in particular, was elevated in both metastatic variants (FIGS. 2and 2). We observed robust levels of CAIX protein localized to theplasma membrane in the metastatic tumors, whereas CAIX expression wasabsent from the non-metastatic 67NR tumors. A Western blot showing CAIXoverexpression in the primary tumors is reproduced in FIG. 3. NMG=normalmammary gland. Beta-actin served as a loading control.

These data indicate that hypoxia-induced CAIX expression may be criticalfor increasing the metastatic potential of primary breast tumors.

Example 4 Validation of the 4T1 Model with Respect to CAIX Expression,pH Regulation and Cell Survival in Hypoxia

For these experiments, 4T1 cells were cultured for 48 h in normoxia orhypoxia and the levels of CAIX expression were analyzed using qRT-PCRand Western blots with beta-actin acting as a control. Data areexpressed as mean±s.e.m. n=3, **P<0.005, ***P<10⁻³. In a second step,4T1 cells expressing non-silencing shRNA (shNS) or shRNA targeting CAIX(shCAIX) were incubated for 72 h. Two independent clones (C2, C5)expressing shCAIX were analyzed. Data are expressed as means±s.e.m. n=3.***P<0.0005, compared to cells cultured in normoxia. CAIX geneexpression in the 4T1 cells was silenced by stably expressing constructstargeting mouse CAIX and cultured the cells in hypoxia to determine theefficacy of the shRNA to inhibit hypoxia-induced expression of CAIX.

Thus, metastatic cell lines, especially the 4T1 cell line, induced CAIXexpression in response to hypoxia (FIG. 4A). In contrast, culture inhypoxia did not induce CAIX expression in the non-metastatic cell line67NR. 4T1 Cells expressing a non-silencing control shRNA (shNS)dramatically upregulated the expression of CAIX in hypoxia (FIG. 4B),whereas hypoxia-induced CAIX expression was markedly attenuated in twoindependent clones (C2, C5) expressing shRNA targeting CAIX (shCAIX;FIG. 4B).

CAIX is functionally linked to the control of tumor pH through itsregulation of the intracellular and extracellular pH. Hypoxia-inducedextracellular acidosis is a measure of the biological activity of CAIX.Acidification of the extracellular medium in hypoxia is blocked in theshCAIX-expressing 4T1 clones relative to the parental and shNSexpressing 4T1 cells, suggesting that silencing CAIX gene expressioninduces functional inhibition of pH regulation in the metastatic 4T1cells.

4T1 cells depleted of CAIX showed increased cell death compared tonon-silencing control cells when cultured in hypoxia (FIG. 5). Thissuggests that CAIX is important for the survival of metastatic breastcancer cells in hypoxic environments.

Example 5 In Vivo Demonstration

Stable depletion of CAIX in the 4T1 mouse breast tumor model inhibitsprimary tumor growth and metastasis as shown in FIG. 6. Specifically,representative bioluminescent images of spontaneous metastasis using the4T1 tumor model are used to demonstrate this finding. Pseudo-color heatmaps (light, least intense, dark, most intense) are shown overlaid onmurine body images. For these studies, 4T1 cells expressing shNS orshCAIX and parental 4T1 cells were inoculated into the mammary gland ofBALB/c mice. Animals were monitored for tumor growth. n=10 for eachgroup. Results are expressed as means±s.e.m. * denotes completion ofprimary tumor excision from the control groups. ***P<10⁻¹¹ with atwo-sided Student's t-test, compared to the shNS group.

The results show that 4T1 cells readily form tumors that grow steadilyover 30 days while tumors established from CAIX-depleted cells regressedsignificantly after initial tumor growth (FIG. 6A). The regression ofthe tumors appeared to be stable, as there are only two mice withprimary tumor recurrence appearing towards the end of the study (FIG.6B). Thus, elimination of CAIX expression has a dramatic effect on theoverall survival of the mice. While the animals bearing tumors thatexpress CAIX have to be sacrificed due to progressive metastaticdisease, the survival rate of animals inoculated with CAIX-depleted 4T1cells remained at 100%.

CAIX expression is down regulated in the tumors derived fromCAIX-depleted 4T1 cells. There is no difference in the expression levelsof CAXII between the shCAIX 4T1 tumors and the control tumors,suggesting that tumor growth suppression in this model occurs in thepresence of CAXII, and that CAIX is the critical enzyme for survival andgrowth of hypoxic breast tumors.

Example 6 Breast Tumor Models

Stable depletion of CAIX in the 4T1 mouse breast tumor model inhibitsprimary tumor growth and metastasis. In FIG. 6(A) spontaneous metastasisusing the 4T1 tumor model are visualized using bioluminescence heat maps(lighter, least intense, darker, most intense) overlaid on gray-scalebody images. (B) 4T1 cells expressing shNS or shCAIX and parental 4T1cells were inoculated into the mammary gland of BALB/c mice. Ten animalsin each group were monitored for tumor growth. Arrows denote changes inthe number of animals, and revised values are indicated. The results areexpressed as means±s.e.m. A “*” denotes completion of primary tumorexcision from the control groups. ***P<10⁻¹¹ with a two-sided Student'st-test, compared to the shNS group.

CAIX depletion in human breast cancer MDA-MB-231 cell line with CAIXshRNA shows significant inhibition of hypoxia-induced CAIX expression inthese cells relative to parental and non-silencing control cells (FIG.7). Moreover, depletion of CAIX dramatically attenuates tumor growth ofMDA-MB-231 xenografts.

FIG. 7 illustrates the evidence that stable depletion of CAIX in a humanbreast tumor model inhibits primary tumor growth. MDA-MB-231 cellsexpressing shNS or shCAIX and parental MDA-MB-231 cells weresubcutaneously inoculated into flank of NOD.CB17-prkdc^(scid)/J mice andanimals were monitored for tumor growth (n=7 for each group). *P<0.01with a two-sided Student's t-test, compared to shNS control tumors. Inthe FIG. 7 inset, MDA-MB-231 cells expressing shRNAmir targeting humanCAIX (shCAIX) or a non-silencing control sequence (shNS) were culturedin normoxia or hypoxia for 72 h and analyzed for hypoxia-induced CAIXexpression. Western blot is shown. β-actin served as a loading control.

Example 7 Metastases Model

Cell line 4T1 injected intravenously form robust lung metastases andsubject mice had to be euthanized within 3 weeks post injection due tometastatic progression, but no metastases were observed in mice that hadbeen inoculated with the CAIX-depleted cells (FIG. 8A). Tumor cellsdepleted of CAIX showed almost no visible metastasis to the lungs andremain completely healthy (FIG. 8B). Negative control 67NR cells, whichare not spontaneously metastatic, show little evidence of metastasisafter three weeks post-injection, despite the fact that the cellsconcentrated in the lungs at 24 hours post-injection.

Examination of lungs from animals injected with cells expressing CAIXexhibited large numbers of lung surface nodules, while the lungs frommice injected with cells depleted of CAIX were essentially normal (FIG.8C). Stable depletion of CAIX inhibits establishment of lung metastasesin the 4T1 model.

Membrane-localized CAIX expression was evident in histologic sections oflungs from control animals, but not from mice bearing CAIX-depletedcells (FIG. 8D).

Example 8 Selectivity of MST-017 (CAI 17)

Cells were cultured for 72 h in the presence of 10 μM MST-017. Shown inFIG. 9 are representative images of the FITC-tagged inhibitor bound tothe cell lines in the indicated conditions. (C) Cells were cultured for72 h with or without MST-017 (400, 600 and 400 μM for the 4T1, 66cl4 and67NR cells, respectively). n=3. The mean changes in extracellularpH±s.e.m. are shown. For each cell line, changes in the extracellular pHin hypoxia were assessed relative to the baseline extracellular pHmeasured in parallel cultures grown in normoxia. *P<0.001 with atwo-sided Student's t-test, compared to cells cultured withoutinhibitor.

The extracellular pH decreased dramatically in hypoxia in the 66cl4 andthe 4T1 metastatic cell lines which express CAIX, but remained unchangedin the 67NR cultures which do not express CAIX. Treatment of the cellswith MST-017 reversed acidification of the extracellular medium underhypoxia in the 66cl4 and 4T1 cell cultures (FIG. 9C).

Example 9 In Vivo Tumor Inhibition Using Sulfonamide Compound MST-017

Targeting CAIX activity with a specific small molecule inhibitorattenuates the growth of 4T1 primary tumors (FIG. 10). In FIG. 10(A),4T1 Cells were cultured in normoxia or hypoxia and levels of CAIXexpression were analyzed by Western blot.

Animals were inoculated orthotopically with 4T1 cells. Tumors wereallowed to establish for 14 days. Mice were injected with the indicateddoses of MST-017 3× per week for 2 weeks. Left panel, tumor growth wasmonitored by caliper-based measurement. Treatment initiation andtermination are indicated by arrows (FIG. 10B). Vehicle-treated anduntreated animals served as controls. *P<0.02, **P<0.01 using a 2-sidedStudent's t-test, compared to vehicle controls.

The weights of treated animals were monitored as a measure of generalinhibitor toxicity. Mice were weighed just prior to each dose of theCAIX inhibitor. No significant differences in the weights among thevarious treatment and control arms were noted (FIG. 100).

Thus, treatment of mice harboring established 4T1 tumors with MST-017showed significant inhibition of tumor growth in treated mice comparedto vehicle controls. The inhibitor concentrations and the dosingschedule were well-tolerated, as no significant weight reduction wasnoted in the treated mice.

Treated mice harboring established 67NR-derived tumors with identicalconcentrations of MST-017 showed no significant effect of the inhibitorrelative to the vehicle control (FIG. 11) and no significant weightreduction was observed.

Example 10 In Vivo Metastases Inhibition with Novel Sulfonamides MST-104and MST-119

Novel CAIX inhibitor MST-119 reduces the formation of metastases by 4T1mammary tumor cells. The chemical structure of CAIX inhibitor MST-119 isshown in FIG. 12A. Representative bioluminescent images of metastasesestablished following intravenous injection of 4T1 cells and treatmentwith MST-119 is shown in 12B. Animals were treated 24 hours postinoculation of cells. Three doses were administered by i.p. injectionover 6 days and the mice were imaged 24 hours following the third doseof inhibitor. MST-119 was delivered in a vehicle comprised of 37.5%PEG400, 12.5% ethanol and 50% saline. Quantification of tumor-derivedbioluminescence is shown in 12C. Regions of interest were positionedaround metastatic foci and total flux (photons/sec) at the mouse surfacewas calculated. Data are reported as the mean±s.e.m. N=4 per group.*P<0.05.

Novel CAIX inhibitor MST-104 reduces formation of metastases by 4T1mammary tumor cells (FIG. 13). 4T1 cells were injected directly into thetail vein of BALB/c mice. Daily treatment for 5 days with vehicle orMST-104 was initiated 24 hours post inoculation of cells and mice wereimaged 24 hours following the final dose. Vehicle and inhibitor wereadministered by i.p. injection. Shown are representative images oftumor-cell derived bioluminescence in control and inhibitor-treatedanimals. (B) The graph shows quantification of tumor-derivedbioluminescence. n=6 per group. *P<0.01. Quantification of thebioluminescent signal revealed a statistically significant decrease inthe formation of metastases in the treated mice. These data providefurther illustration of the inhibition of formation of lung metastasesby breast cancer cells in response to targeted inhibition of CAIX usingnovel sulfonamides.

Example 11 Inhibition of Human Primary Breast Tumor Growth with NovelSulfonamides

Novel CAIX inhibitor MST-104 reduces the growth of human primary breastcancer xenografts. Metastatic MDA-MB-231 LM2-4^(Luc+) cells wereimplanted orthotopically into NOD/SCID mice. When tumors reached anaverage of 200 mm³, animals received the indicated doses of MST-104daily by i.p. administration and tumor growth was quantified usingcaliper measurements. The initiation and termination of inhibitortreatment is indicated. n=8/group. *P<0.03, **P<0.001. Inset, Westernblot showing CAIX expression by the LM2-4^(Luc+) cells cultured innormoxia (N) and hypoxia (H).

To evaluate the effect of pharmacologic inhibition of CAIX activity invivo, we treated mice harboring established MDA-231 LM2-4 tumors (Eboset al., 2009) with MST-104. These cells were observed to induce robustlyCAIX in hypoxia (FIG. 14, inset). We observed significant,dose-dependent inhibition of tumor growth in mice treated with theinhibitor, compared to vehicle controls (FIG. 14). These data show theability of sulfonamide-based CAIX inhibitors to specifically targetCAIX-expressing human breast tumors.

Example 12 Inhibition of Hypoxia-Induced Invasion and Survival of HumanBreast Cancer Cells Grown in 3D Matrigel™ Cultures by Novel Sulfonamides

MDA-MB-231 LM2-4^(Luc+) cells grown in 3D Matrigel™ cultures areinvasive in hypoxia (FIG. 15). Cells were cultured in a 3D “on-top”Matrigel™ assay for 4 days in normoxia and hypoxia as described in themethods. Representative phase-contrast images of 3D cultures are shown.Hypoxia induced invasion by the LM2-4 variant, but not by the parentalMDA-231 cells.

Treatment with novel sulfonamide inhibitors of CAIX attenuateshypoxia-induced invasion of human breast cancer cells grown in 3DMatrigel™ cultures (FIG. 16). Cells were cultured in 3D “on-top”Matrigel™ assays for 4 days in hypoxia in the presence of inhibitors.DMSO-treated cell cultures served as controls. The percentageconcentration of DMSO and molar concentration of inhibitors areindicated. Representative phase contrast images are shown. These datashow that sulfonamides inhibit invasion of metastatic human breastcancer cells in hypoxia.

Ureido sulfonamide inhibitors of CAIX show differential effects on celldeath of human breast cancer cells in hypoxia (FIG. 17). Cells werecultured in 3D “on-top” Matrigel™ assays. Cells were growth in thepresence of inhibitors for 4 days in hypoxia. DMSO-treated cell culturesserved as controls. (A)

Representative images of TUNEL-positive cells (arrows). (B) Graphshowing quantification of TUNEL +ve cells by counting 5 random fieldsper condition. Data are expressed as the average number ofTUNEL-positive cells/20× field of view (FOV). These data show thatsulfonamide inhibitors of CAIX can induce death of human breast cancercells in hypoxia.

Example 13 Genetic Depletion of CAIX Expression in Breast Cancer CellsReduces the Cancer Stem Cell Population in Hypoxia

In vitro proliferation in suspension under serum free conditions asnon-adherent tumorspheres is a characteristic of breast cancer stemcells. The breast cancer stem cell population has previously beencharacterized as displaying the CD44⁺CD24^(−/low) signature (Al-Hajj etal, (2003) Proc Natl Acad Sci USA 100:3983-3988); Ponti et al, (2005)Cancer Res 65:5506-5511). FIG. 18 shows that CAIX expression is requiredfor growth of the “tumorsphere initiating” population andtumorsphere-forming efficiency in hypoxia. (A) 4T1 shNS and shCAIX cellswere seeded at doubling dilutions and cultured under tumorsphere-formingconditions in normoxia or hypoxia. The number of cells required toinitiate tumorsphere growth was assessed. Mean±SEM of three independentexperiments is shown. *P<0.03, **P<0.006. (B) 4T1 shNS and shCAIX cellswere cultured as tumorspheres in normoxia or hypoxia, disaggregated andthe CD44+CD24−/low population assessed by FACS analysis. Data shown arethe mean changes in % CD44+CD24−/low cells±SEM, from 3 independentexperiments. *P<0.004, **P<0.025.

No significant difference in the number of cells required to formtumorspheres was observed between 4T1 shNS and shCAIX in normoxia, sinceCIAX is not induced in either cell line at normal oxygen levels (FIG.18A). The number of 4T1-shNS cells required to form tumorspheres wassignificantly reduced in hypoxia, compared to normoxia controls,suggesting that the percentage of cancer stem cells (CSC) issignificantly higher in hypoxic cultures (FIG. 18A). Importantly,RNAi-mediated knock down of CIAX expression significantly increased thenumber of seeding cells required to form a tumorsphere in hypoxia,showing that CAIX expression is required for the observed CSC-likeexpansion in hypoxia with shNS-4T1 controls cells.

In FIG. 18B, 4T1 shNS and shCAIX cells were cultured under tumorsphereforming conditions, in normoxia or hypoxia, and analyzed by FACS toquantify the putative CSC-like population labeled as CD44⁺CD24^(−/low).In shNS controls, the CD44⁺CD24^(−/low) population is significantlyincreased in hypoxic culture conditions, compared to normoxic controls(FIG. 18B). However, RNAi-mediated knockdown of CAIX significantlydepletes this population in hypoxia, showing that CAIX is required forCD44⁺CD24^(−/low) CSC-like expansion in hypoxia (FIG. 18B).

Example 14 MST-104 can Deplete the Cancer Stem Cell Population in HumanBreast Orthotopic Tumors In Vivo

FIG. 19 shows that treatment of human primary breast cancer xenograftswith CAIX inhibitor MST-104 targets the cancer stem cell population invivo. MDA-MB-231 LM2-4^(luc+) were implanted orthotopically intoNOD/SCID mice. When tumors reached an average of 200 mm², animalsreceived either vehicle or 38 mg/kg MST-104 daily by i.p.administration. (A) Primary tumors were removed, dissociated and ESA+cell population assessed by FACS analysis. Representative FACS plotsdemonstrating the percentage of ESA+ cells are shown. (B) Data shown arethe mean changes in ESA+ cells±SEM, from 3 mice. **P<0.0224.

Vehicle treated tumors contained a mean 10% ESA+ cell population. Incomparison, tumors treated with sulfonamide CAIX inhibitor MST-104 alsodisplayed a significantly reduced ESA+ cell population, compared tovehicle-treated control tumors. These data show that sulfonamides candeplete the human breast cancer stem cell population in vivo.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

While specific embodiments have been described and illustrated, suchembodiments should be considered illustrative only and not as limitingthe invention as construed in accordance with the accompanying claims.

1. A pharmaceutical composition comprising:4-{[(4′-Fluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-104) or4-{[(3′-Nitrophenyl)carbamoyl]amino}benzenesulfonamide (MST-119); and apharmaceutically acceptable excipient.
 2. A pharmaceutical compositionaccording to claim 1 wherein the compound is4-{[(3′-Nitrophenyl)carbamoyl]amino}benzenesulfonamide (MST-119).
 3. Apharmaceutical composition according to claim 1 wherein the compound is4-{[(4′-Fluorophenyl)carbamoyl]amino}benzenesulfonamide (MST-104).